Miniaturized Microparticles

ABSTRACT

An encoded microparticle having a spatial code is provided; and a set of encoded microparticles possessing subsets each provided with a distinguishable spatial code, wherein the codes comply with a pre-determined coding scheme. Presented are also methods of using the encoded microparticles in various biological assays, such as various multiplex assays and visualizing them by creating a digital image of the encoded microparticles and determining whether false positives are present. Further are provided methods of manufacture of the encoded microparticles which employ ferromagnetic nanoparticles applied using spin-on-glass techniques.

CROSS-REFERENCE TO RELATED CASES

This US patent application is a continuation-in-part of co-pending U.S.patent application Ser. No. 12/779,401 filed May 13, 2010, which is acontinuation of U.S. patent application Ser. No. 11/521,057 filed Sep.13, 2006, now U.S. Pat. No. 7,745,091, which claims priority from U.S.provisional application Ser. No. 60/762,238 filed Jan. 25, 2006 and U.S.provisional application Ser. No. 60/716,694 filed Sep. 13, 2005, thesubject matter of each being incorporated herein by reference in theirentirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:AFFY_(—)61-070US_SeqList_ST25.txt, date recorded: Jun. 30, 2010, filesize 2 kilobytes).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the art of microstructures, and moreparticularly to encoded microparticles, manufacture thereof and uses inassays.

BACKGROUND OF THE INVENTION

Microparticles or nanoparticles are often referred to as structureswhose characteristic dimensions are on the order of micrometers or less,such as those with volumes of 1 mm³ or less. Due to their uniqueproperties arising from their small characteristic dimensions,microparticles have found distinguishable applications in laboratoryresearch and many industrial fields. Encoded microparticles possess ameans of identification and are an important subclass of the generalfield of microparticles. Because encoded particles carry information andcan be physically tracked in space and time, they greatly extend thecapabilities of non-encoded particles. A particularly importantapplication for encoded microparticles is multiplexed bioassays,including those involving DNA and proteins. Other important fields forencoded microparticles include combinatorial chemistry, tagging, etc.Many biochemical and non-biochemical applications as will be discussedherein below.

For many applications, one more desirable attributes include: a largenumber of identifiable codes (i.e. a high codespace), accurate andreliable identification of the encoded particles, material compatibilityfor a particular application, low cost manufacturing of themicroparticles (on a per batch, per particle, and per code set basis),and flexibility in the detection systems.

Several approaches to produce encoded microparticles have been developedin the past, such as fragmented colored laminates, colored polystyrenebeads, quantum dot loaded polymer beads, rare-earth doped glassmicrobarcodes, electroplated metal nano rods, diffraction grating basedfiber particles, and pattern bars and disks, and other types ofmicroparticles. These technologies however suffer from any of a numberof limitations, such as, insufficient codespace, high cost, inadequateprecision, poor performance in applications, problematic clumpingincapability of large scale manufacture, and complicated preprocessingor assay procedures.

Therefore, what is desired is an encoded microparticle or a set ofencoded microparticles carrying coded information, methods of making thesame, methods for providing the codes for microparticles, methods forfabricating the microparticles, methods and systems for detectingmicroparticle, and methods and systems for using.

SUMMARY OF THE INVENTION

As an example of the invention, an encoded microparticle is disclosedherein. The microparticle comprises: a longest dimension less than 50μm; an outer surface substantially of glass; and a spatial code that canbe read with optical magnification.

Embodiments presented include methods of detecting the encodedmicroparticles using software algorithms and microscopy techniques.

Other embodiments include encoded microparticles havingsuperparamagnetic properties through the use of ferromagnetic nanobeadsimbedded into the encoded microparticles using spin-on-glass techniques.

BRIEF DESCRIPTION OF DRAWINGS

While the appended claims set forth the features of the presentinvention with particularity, the invention, together with its objectsand advantages, may be best understood from the following detaileddescription taken in conjunction with the accompanying drawings ofwhich:

FIG. 1A schematically illustrates an encoded microparticle of theinvention;

FIG. 1 b is a side view cross-section of the microparticle in FIG. 1 a;

FIG. 2 schematically illustrates another example encoded microparticleof the invention;

FIG. 3 a schematically illustrates another example encoded microparticleof the invention;

FIG. 3 b schematically illustrates an another example encodedmicroparticle of the invention;

FIG. 4 a and FIG. 4 b schematically illustrates an exemplarymicroparticle whose coding structures are derived from a singlematerial;

FIG. 4C schematically illustrates an another example encodedmicroparticle of the invention;

FIG. 4 d is a cross-sectional view of another exemplary microparticleduring an exemplary fabrication of the invention;

FIG. 5 is a flow chart showing the steps executed in an exemplaryfabrication method of the invention;

FIG. 6 a to FIG. 6 m are cross-section views and top views of amicroparticle in an exemplary fabrication process of the invention;

FIG. 7 is a cross section view of a microparticle from the exemplaryfabrication process in FIG. 6 a to FIG. 6 m.

FIG. 8 is a perspective view of an array of microparticles on asubstrate during the fabrication;

FIG. 9 a to FIG. 10 are SEM images of a plurality of microparticlesduring the fabrication of an exemplary fabrication method of theinvention;

FIG. 11 a and FIG. 11 b illustrate an exemplary etching method that canbe used in the fabrication method of the invention;

FIG. 12 a and FIG. 12 b are images of a plurality of microparticles ofthe invention;

FIG. 13 a to FIG. 13 c schematically illustrate an exemplary wafer levelfabrication method according to an exemplary fabrication method of theinvention;

FIG. 14 presents a reflectance-mode inverted microscope image of 8encoded microparticles of the present inventions;

FIG. 15 shows a diagram of an optical system used to image the encodedmicroparticles of the invention;

FIG. 16 presents a full field, single image taken at the samemagnification as that in FIG. 14;

FIG. 17 shows a high magnification image of encoded microparticles;

FIG. 18 a shows a montage of 12 dense reflectance images of encodedmicroparticles;

FIG. 18 b shows a transmission fluorescence microscope image of examplemicroparticles of the invention;

FIG. 19A shows a full field reflectance image;

FIG. 19B shows the same image selection of FIG. 19A after the imageprocessing to associate discrete segments into full microparticles;

FIG. 20A shows a selection of a reflectance image;

FIG. 20B shows the same image selection of FIG. 20A after the imageprocessing to associate discrete segments into full microparticles;

FIG. 21 illustrates a processed image is shown on the right and pixelintensity profiles from 4 example microparticles are shown on the left;

FIG. 22 shows a schematic of a specially prepared surface that havefeatures designed to immobilize and separate the encoded microparticlesfor imaging;

FIG. 23 and FIG. 24 show a flow-cell enabling the microparticles flowingin a fluid can be provided for detection by continuous imaging;

FIG. 25 illustrates another alternative microparticle of the invention;

FIG. 26 shows a diagram of a spatially optically encoded microparticlewith a fluorescent outer layer;

FIGS. 27 a to 27 c show schematic diagrams of encoded microparticles ofthe present invention with surface indentations that form a spatialcode;

FIG. 27 d shows an example of encoded microparticles comprisingindentations;

FIGS. 28 a to 28 c show the non-uniform aerial density measured normalto the particle surface for corresponding particles in FIGS. 27 a to 27c;

FIG. 29 a to FIG. 30 c are top views of microparticles according toanother example of the invention during another exemplary fabrication ofthe invention;

FIG. 31A to 31C show drawings of the 3 mask fields of the preferredembodiment of the microparticle structure and FIG. 31D shows a drawingof a reticle plate;

FIG. 32 shows an alternate example of the general method of generatingcode using multiple print steps utilizes stamping;

FIG. 33A to FIG. 33M illustrate the microfabrication process steps ofthe example encoded microparticle of FIG. 1A;

FIG. 34 a to FIG. 34 m show the corresponding cross sectional views ofthe microparticle in FIG. 33 a to FIG. 33 m;

FIG. 35A to FIG. 35 c show exemplary microparticles that can be producedusing the method of the invention;

FIG. 36 shows four microscope images of actual encoded microparticles,just prior to release from the dies;

FIG. 37 shows charts of example data that is input into the steppersoftware to generate different codes on every die on a wafer;

FIG. 38 shows drawings of an example scheme for producing an increasednumber of codes per die;

FIG. 39A shows a graphical representation of encoded microparticles thatare formed according to the invented non-binary coding scheme;

FIGS. 39B and 39C show random codes with different numbers of gaps andgaps of varying location;

FIG. 40 shows photographs a montage of 4 photographs of various forms ofa large prototype set of microparticles;

FIG. 41 is a flow chart of an exemplary bioassay process;

FIG. 42 shows a diagram of an example of the process by which wholewafers become mixtures of particle-probe conjugates that are ready to bereacted with samples to perform a bioassay;

FIG. 43 shows a diagram of an optical system used to image encodedmicroparticles that utilizes two CCD cameras for the simultaneousacquisition of a reflectance and fluorescence image;

FIGS. 44 and 45 show dense fluorescence microscope image of amultiplicity of encoded microparticles;

FIG. 46A and FIG. 46B show a reflectance and fluorescence image pair forthe same set of microparticles of the invention; and 46C is an overlayof the images in FIG. 46A and FIG. 46B;

FIG. 47A to FIG. 47F show dense fluorescence microscope images ofencoded microparticles in a time sequence;

FIG. 48 shows real assay data from a 2-plex DNA hybridization assay;

FIG. 49 a illustrates an exemplary assay in which the microparticles ofthe invention can be used;

FIG. 49 b illustrates another exemplary assay in which themicroparticles of the invention can be used;

FIG. 50 illustrates another exemplary assay in which the microparticlesof the invention can be used;

FIG. 51 is a schematic that includes images of particles but is not theresult of an actual experiment of this invention; and

FIGS. 52A to 52C show flowcharts of examples of the code elementpatterning and etch steps.

FIG. 53 provides a schematic of the algorithm operation.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

An encoded microparticle is provided carrying a code, and a set ofencoded microparticles are provided with distinguishable codes, whereinthe codes comply with a pre-determined coding scheme. Preferably, themicroparticles in the examples below have a volume of 1 mm³ or less. Themicroparticle of the invention enables fast, precise and lesscomplicated detection of the code. Methods for providing the codes onmicroparticles, methods for fabricating the microparticles, methods andsystems for detecting the microparticle, and methods and systems forusing the microparticles are also disclosed.

In the following, the invention will be discussed with reference tospecific examples. It will be appreciated by those skilled in art thatthe following discussion is for demonstration purposes, and should notbe interpreted as a limitation. Instead, other variations withoutdeparting from the spirit of the invention are also applicable.

Overall Structure of the Microparticle

As an example, FIG. 1A schematically illustrates an encodedmicroparticle of the invention. Microparticle 100 is a cuboid structureelongated along the Y direction in the Cartesian coordinate as shown inthe figure. The cross-sections perpendicular to the length of themicroparticle have substantially the same topological shape—which issquare in this example.

The microparticle in this particular example has a set of segments (e.g.segment 102) and gaps (e.g. gap 104) intervening the segments.Specifically, segments with different lengths (the dimension along thelength of the microparticle, e.g. along the Y direction) representdifferent coding elements; whereas gaps preferably have the same lengthfor differentiating the segments during detection of the microparticles.The segments of the microparticle in this example are fully enclosedwithin the microparticle, for example within body 106. As an alternativefeature, the segments can be arranged such that the geometric centers ofthe segments are aligned to the geometric central axis of the elongatedmicroparticle. A particular sequence of segments and gaps represents acode. The codes are derived from a pre-determined coding scheme.

Segments of the microparticle can be any suitable form. In an example ofthe invention, each segment of the microparticle has a substantiallysquare cross-section (i.e. the cross-section in the X-Z plane of aCartesian coordinate as shown in FIG. 1A) taken perpendicular to thelength (i.e. along the Y direction in the Cartesian coordinate in FIG.1A) of the microparticle. The segments may or may not be fabricated tohave substantially square cross-section. Other shapes, such asrectangular, circular, and elliptical, jagged, curved or other shapesare also applicable. In particular, the code elements—i.e. segments andgaps, may also take any other suitable desired shape. For example, thesegment (and/or the gaps) each may have a cross-section that isrectangular (e.g. with the aspect ratio of the rectangular being 2:1 orhigher, such as 4:1 or higher, 10:1 or higher, 20:1 or higher, or even100:1 or higher, but preferably less than 500:1).

The microparticle example of FIG. 1A has six major surfaces, namelysurfaces of (X=±x₀, Y, Z), surfaces (X, Y, Z=±z₀), and surfaces (X,Y=±y₀, Z), wherein x₀, y₀, and z₀ are respectively the width, length,and height of the microparticle. According to the invention, at leasttwo of the above six surfaces X=±x₀ (or surfaces Z=±z₀), more preferablyfour of the above six major surfaces X=±x₀, surfaces Z=±z₀ aresubstantially continuous, regardless of whether each surface has or doesnot have indentations. With this configuration, the microparticleexhibits substantially the same geometric appearance and specificproperties to the detector—such as an optical imaging apparatus. Infact, the major surfaces can be made substantially flat. For example,even though roughness or varying profiles may be caused duringfabrication, substantially flat major surfaces can still be obtainedusing standard surface machining techniques, such as over-deposit andetch back or chemical-mechanical-polishing (CMP) techniques, as well asproper control of patterning steps to create smooth vertical sidewallprofiles.

The code elements, i.e. the segments and gaps, may take any desireddimensions. As an example of the invention, each coding structure has acharacteristic dimension that is 5 μm (microns) or less, such as 3microns or less, and more preferably 1 micron or less, such as 0.8 or0.5 microns or less. In particular, when gaps are kept substantially thesame dimension while the segments vary in dimension, each gap preferablyhas a characteristic dimension that is 1.5 microns or less, such as 0.8or 0.5 microns or less.

As one example, if forming the microparticles on a 12-inch silicon waferwith 0.13 line widths, the gap areas can be made to have 0.13 μm minimumwidths, with the less transparent segments having widths of from 0.13 μmto much larger (depending upon the desired length of the particle andthe encoding scheme and code space desired). Minimum gap widths, as wellas minimum segment widths, of from 0.13 to 1.85 μm (e.g. from 0.25 to0.85 μm) are possible depending upon the wafer fabrication used. Ofcourse larger minimum gap and segment lengths (e.g. 1.85 to 5.0 μm, ormore) are also possible. Other sized wafers (4 inch, 6 inch, 8 inchetc.) can of course be used, as well as wafers other than silicon (e.g.glass), as well as other substrates other than silicon (larger glasspanels, for example).

Though the microparticle may have the same length in the X, Y and/or Zdirections, preferably the encoded microparticle has a ratio of thelength to width of from 2:1 to 50:1, e.g. from 4:1 to 20:1. In anexample of the invention, the microparticle has a length (e.g. thedimension along the Y direction) of 70 microns or less, 50 microns orless, 30 microns or less, such as 20 microns or less, 16 microns orless, or even 10 microns or less. The width (e.g. the dimension alongthe X direction), as well as the height (the dimension along the Zdirection), of the microparticle can be 15 microns or less, 10 micronsor less, 8 microns or less, 4 microns or less, or even 1 microns orless, such as 0.13 micron. Widths as small as from 0.5 to 2 microns arealso possible. Other than the shape as shown in FIG. 1A and discussedabove, the microparticle may take a form of rod, bar, disk or any otherdesired shapes.

The coding structures and gaps of the microparticles can take anysuitable form as long as the coding structures and gaps togetherrepresent detectable codes. As mentioned above, the cross-section of themicroparticles, as taken perpendicular to the length of the particle,can be square, rectangular, circular, elliptical, or any desired shapesuch as jagged or curved shapes or other profiles. When thecross-section is rectangular, the rectangle preferably has an aspectratio (the ratio of the length to the width or height) of 2:1 or higher,such as 4:1 or higher, 10:1 or higher, 20:1 or higher, or even 100:1 orhigher, but preferably less than 500:1. The ratio of the width to heightcan be around 1:1 (square cross section), or have a ratio of from 1:4 to1:1—preferably a ratio that allows the particle to rest on either thesides defining the width or height of the particle such that the code ofthe microparticle can be detected regardless of which of the elongatedsides the particle rests.

To facilitate fast, cost-effective, reliable, and easy detection of thecode represented by the coding structures and gaps, it is preferred thateach coding structure is as omni-directional as possible to thedetection means. That is—each coding structure exhibits substantiallythe same geometric appearance or detectable properties when observedfrom at least two directions, more preferably from four (or all, if notfour-sided in cross section) directions perpendicular to the length ofthe microparticle. Accordingly, the coding structures preferably possessrotational symmetry along the length of the microparticle, such as2-folded or 4-folded rotational symmetry.

A microparticle of the invention can have any suitable number of codingstructures depending upon the shape or length of the particle, and thecode space desired. Specifically, the total number of coding structuresof a microparticle can be from 1 to 20, or more typically from 3 to 15,and more typically from 3 to 8.

The desired code can be incorporated in and represented by themicroparticle in many ways. As an example, the coding elements of thepre-determined coding scheme can be represented by the segment(s)—e.g.segments of different lengths represent different coding elements of thecoding scheme. Different spatial arrangements of the segments with thedifferent (or the same) lengths and intervened by gaps representdifferent codes. In this code-incorporation method, the intervening gapspreferably have substantially the same dimension, especially the lengthin the direction to which the segments are aligned. As another example,the codes are incorporated in the microparticle by arranging gaps thatvary in lengths; while the segments have substantially the samedimension and are disposed between adjacent gaps. In another example,the both segments and gaps vary in their dimensions so as to represent acode. In fact, the code can also be represented in many otheralternative ways using the segments, gaps, and the combination thereof.

For representing a code derived from the predetermined coding scheme,the segments and gaps are arranged along the length (the Y direction) ofthe elongated microparticle (2D, or even 3D, arrangements however arealso possible). Specifically, the segments and gaps are alternatelyaligned along the length with the each segment being separated (possiblyfully separated and isolated) by adjacent gaps; and each gap isseparated (possibly fully separated and isolated) by adjacent segments,which is better illustrated in a cross-sectional view in FIG. 1B, whichwill be discussed in the following.

In an example of the invention, any suitable number of segments can beused—e.g. from 2 to 20, or more typically from 3 to 15 segments (moretypically from 3 to 8 segments) of less transparent material (ascompared to the intervening gaps between the segments) are providedwithin the encoded microparticle. To form the code, it is possible thatthe segments of less transparent material are varying lengths.Alternatively, the segments of less transparent material could each havesubstantially the same length whereas the intermediate segments of moretransparent material could have varying lengths. Of course, the segmentsof more transparent material and the intermediate segments of lesstransparent material could both have varying lengths in order torepresent the code.

Referring to FIG. 1B, the cross-section is taken in the Y-Z plane (orequivalently in the X-Y plane) of the particle in FIG. 1A. Segments(e.g. segment 102) and gaps (e.g. gap 104) alternate along the length ofthe microparticle.

In order to enable detection of codes incorporated in microparticles,the segments and gaps in each microparticle can be composed of materialsof different optical, electrical, magnetic, fluid dynamic, or otherdesired properties that are compatible with the desired detectionmethods. In one example the segments and gaps are directly spatiallydistinguishable under transmitted and/or reflected light in the visiblespectrum. For example, when the code detection relies upon opticalimaging, the distinguishable property (segments vs. gaps) can be adifference in transmissivity to the particular light used for imaging(which can be any desired electromagnetic radiation—e.g. visible andnear-visible light, IR, and ultra-violet light. The segments can be madeto be more light absorbing (or light reflecting) than the interveningspacing material (or vice versa). When the code detection relies uponthe electrical property measurements, the property can be resistance andconductance. When the code detection involves magnetic methods, theproperties can be inductance and electro-inductance. When the codedetection involves fluid dynamic methods, the property can be viscosityto the specific fluid used in the code detection. Regardless of whichspecific property is relied upon, the segments and gaps are preferred toexhibit sufficient difference in the specific property such that thedifference is detectable using the corresponding code detection method.In particular, when the code is to be detected by means of opticalimaging, the segments and gaps are composed of materials exhibitingdifferent transmissivity (in an optical transmittance mode) orreflectivity (in optical reflectance mode) to the specific light used inimaging the microparticles. For example, the segments of themicroparticle of the less transparent material can block and/or reflect30% or more, preferably 50% or more, or e.g. 80% or more, of the visiblelight or near visible light incident thereon.

Given the fact that transmissivity of electromagnetic radiation throughan object varies with the thickness of the object, it is preferred thatthe segments that are capable of blocking and/or reflecting 30% or more,preferably 50% or more, or e.g. 80% or more (or even 90% or more), ofthe detection light; while the gaps between the coding structures areprovided from materials and at dimensions that are capable oftransmitting 50% or more, 70% or more, 80% or more, or even 90% or moreof the detecting light. Alternatively, the segments and gaps arecomposed of different materials such that the ratio of thetransmissivity difference is sufficient to detect the code, e.g. is 5%or more, 10% or more, 20% or more, 50% or more, and 70% or more. Thetransmissivity is defined as the ratio of the light intensities of thepassed light to the incident light.

The microstructure can be made of organic and/or inorganic materials ora hybrid of organic and inorganic material. Specifically, the gaps(which are preferably more transmissive to visible or near-visiblelight) and segments (which are preferably less transmissive to visibleor near-visible light as compared to gaps) each can be composed organicor inorganic materials, or a hybrid organic-inorganic material. Thesegments can be composed of a metal (e.g. aluminum), an early transitionmetal (e.g. tungsten, chromium, titanium, tantalum or molybdenum), or ametalloid (e.g. silicon or germanium), or combinations (or nitrides,oxides and/or carbides) thereof. In particular, the segments can becomposed of a ceramic compound, such as a compound that comprises anoxide of a metalloid or early transition metal, a nitride of a metalloidor early transition metal, or a carbide of a metalloid or earlytransition metal and mixtures thereof. Early transition metals are thosefrom columns 3b (Sc, Y, Lu, Lr), 4b (Ti, Zr, Hf, Rf), 5b (V, Nb, Ta,Db), 6b (Cr, Mo, W, Sg) and 7b (Mn, Tc, Re, Bh) of the periodic table.However, preferred are early transition metals in columns 4b to 6b, inparticular tungsten, titanium, zirconium, hafnium, niobium, tantalum,vanadium and chromium.

The gaps which are in this example more transparent, can comprise anysuitable material that is more transparent than the segments. Thespacing material can be a siloxane, siloxene or silsesquioxane material,among others, if a hybrid material is selected. The spacing material, ifinorganic, can be a glass material. Thin film deposited silicon dioxideis a suitable material, with or without boron or phosphorousdoping/alloying agents. Other inorganic glass materials are alsosuitable such as silicon nitride, silicon oxynitride, germanium oxide,germanium oxynitride, germanium-silicon-oxynitride, or varioustransition metal oxides for example. A spin on glass (SOG) could also beused. If an organic material is used for the gap material, a plastic(e.g. polystyrene or latex for example) could be used. Specifically, anSOG layer may be added which has embedded therein other materials, suchas ferromagnetic nanobeads to add superparamagnetic properties to theparticle.

Both the segments and the gaps can be deposited by any suitable methodssuch as CVD (chemical vapor deposition), PVD (physical vapordeposition), spin-on, sol gel, etc. If a CVD deposition method is used,the CVD could be LPCVD (low pressure chemical vapor deposition), PECVD(plasma enhanced chemical vapor deposition), APCVD (atmospheric pressurechemical vapor deposition), SACVD (sub atmospheric chemical vapordeposition), etc. If a PVD method is used, sputtering or reactivesputtering are possible depending upon the desired final material. Spinon material (SOG) or hybrid organic-inorganic siloxane materials mayalso be utilized.

As a more specific example, the segments can be comprised of anysuitable silicon material such as CVD (chemical vapor deposition)deposited amorphous silicon. Polysilicon or single crystal silicon areaalso suitable as are a wide range of other materials as mentioned above.It is preferred, but not necessary, that the material selected for thesegments has a high degree of deposition thickness control, low surfaceroughness, control of etching—both patterning and release (e.g. using adry plasma etch for patterning and a wet or dry chemical etch forrelease), and CMOS process compatibility. The gap material can be CVDdeposited silicon dioxide. The silicon dioxide may includedoping/alloying materials such as phosphorous or boron. Temperatureconsiderations may be taken into account in choosing a combination ofmore and less transparent materials for the segments and gaps.

FIG. 2 schematically illustrates another example encoded microparticleof the invention. Particle 20 has a rectangular cross section and is ofa substantially flat shape. For example the ratio of the height to thewidth of the microparticlecan be any desired ratio, e.g can be from1:1.2 to 1:4 or more, etc.

FIG. 3 a schematically illustrates another example encoded microparticleof the invention. Referring to FIG. 3 a, microparticle 116 is composedof a 1^(st) material 118 and 2^(nd) material 120. The two materials canbe chemically different or have the same chemical composition but bedifferent in another respect such as grain structure or thickness. Thetwo materials are distinguishable with the desired detection scheme. Inthis example each material preferably fully traverses the cross sectionof the particle. An example process for creating this structure involvesfabrication methods as described, including those from the IC/MEMS(Integrated Circuit/Micro-Electro-Mechanical Systems) fields, includingvariations on the patterning and etching methods disclosed herein below,and/or with high energy ion implantation.

FIG. 3 b schematically illustrates another example encoded microparticleof the invention. Referring to FIG. 3 b, the microparticle is comprisedof alternating segments of two different materials 40 and 42 that aresurrounded by a third material 44, whereby the pattern of alternatingsegments forms a detectable code. Other example microparticles maycontain more than two different materials in the interior of theparticle. The particle may have any suitable cross sectional shape andin the example shown, is elongated.

In the examples as discussed above, the microparticle is composed ofmaterials of selected distinguishable properties, such asdistinguishable optical properties. In the example above, one materialhas a greater transparency or optical transmissivity than the othermaterial, which difference is detectable under magnification. A specificexample of the above is where one material is a light absorbingmaterial, and the other material is a translucent or transparentmaterial with greater light transmittance in the visible spectrum (or inanother spectrum should a different detection system be used—e.g. UV, IRetc). In another example, one material is a light reflecting materialwhereas the other material is either light absorbing or lighttransmitting. A detectable difference where one material is more opaqueand the other material is less opaque, or where one material is morereflective and the other material is less reflective, are within thescope of this example. As mentioned above, the alternating portions ofopaque and transparent materials can be made of silicon and glass amongother materials. Given the fact that transmissivity (and reflectivity)of almost all materials exhibit dependencies from the thickness of thematerial, the microparticle may be formed such that the codingstructures (i.e. the structures representing coding elements of a code)are derived from a single material. FIG. 4 a and FIG. 4 b schematicallyillustrates an exemplary microparticle whose coding structures arederived from a single material, such as silicon.

Referring to FIG. 4 a wherein a cross-sectional view of an exemplarymicroparticle is illustrated therein. Microparticle 206 comprises a setof coding structures (e.g. 210, 212, 208, and 214), the combination ofwhich represents a code derived from a coding scheme. For incorporatingthe code, the coding structures have different profiles, such as widthswhile different structures with different widths are positioned atparticular locations. For defining the coding structure and codedetection afterwards, a set of gaps (e.g. gaps 212 and 214) withthicknesses less than the transmissivity threshold thickness (thethreshold below which the material is visible to the particular lightsuch as visible and near-visible light). Different from the example asshown in FIG. 1 a, the coding structures are not fully separated orisolated. The code incorporated in the microparticle can be read basedon the different transmissivity of the coding structure (e.g. 210 and208) which, for example, are less transmissive than the adjacent gaps(e.g. 212 and 214) between the coding structures.

For facilitating the application of the microparticles, especiallybiological/biochemical/biomedical/biotechnology applications wherein thesample bio-molecules are to be attached to the surfaces of themicroparticles, an immobilization layer may be desired to be coated onthe surfaces of the microstructures.

FIG. 4 b schematically illustrates a transmissive-mode image of themicroparticle in FIG. 4 a. Referring to FIG. 4 b, dark regions 210, 208respectively correspond to the coding structures 210 and 208 in FIG. 4a. White regions 212 and 214 respectively correspond to the codingstructures 212 and 214 in FIG. 4 a. Even though the material used in themore light transmitting and less light transmitting sections is thesame, the transmittance profile can still allow for a detectable code.Such a microparticle in FIG. 4A can be formed with a bottom layer ofanother material (e.g. silicon dioxide), and be coated with a secondlayer of another material (e.g. silicon dioxide) if desired. Such amicroparticle can also be fully encased in a material (e.g. silicondioxide) such that it has substantially the same rectangular parallelpiped shape as the structure in FIG. 1 a. FIG. 4C schematicallyillustrates another example encoded microparticle of the invention.Referring to FIG. 4C, the microparticle comprises larger regionsconnected by narrower regions. The microparticle is surrounded by amaterial such that a code is detectable.

The microparticle of FIG. 4A and FIG. 4C can be fabricated in many ways,one of which is schematically demonstrated in a cross-sectional view ofthe microparticle during the exemplary fabrication in FIG. 4D. Referringto FIG. 4D, substrate 216 composed of material (e.g. glass, quartz, orother suitable materials) that is transmissive to a particular light(e.g. visible or near-visible light) is provided. Detaching layer 217 isdeposited on substrate 216. The detaching layer is provided fordetaching the microparticles from the glass substrate afterward byetching or other suitable methods. The etching can be wet, dry, orplasma etching; and the detaching layer is thus desired to be composedof a material etchable with the selected etching method, as discussedhereinabove. As described for previous embodiments of the particlestructures, the detaching layer may be omitted such that the particle isformed directly on the substrate and is subsequently released by a bulketch of the substrate.

A coding structure layer is deposited and patterned so as to form thecoding structures, such as structures 218, 222, 220, 224. After formingthe coding structures, surrounding layer 224 is deposited on the formedcoding structures. Because the surrounding layer will be exposed to thetarget sample in the assay, it is desired that layer 224 is composed ofa material that is resistant to chemical components in the assaysolution wherein the microparticles are to be dispensed. Moreover, forholding the probe molecules, such as nucleic acids (e.g. DNA or RNA),proteins, antibodies, enzymes, drugs, receptors, or ligands, moleculeson the surface of the layer, layer 224 is desired to be capable ofimmobilizing the probe molecules.

Fabrication Process

The following exemplary fabrication processes will be discussed inreference to microparticles with segments and gaps, however it should benoted that the following methods are applicable to many other types ofcode elements.

The microstructure of the invention can be fabricated with a method thatfall into the broad field of micro-machining, such as MEMS fabricationmethods. MEMS use the techniques of the semiconductor industry to formmicroscale structures for a wide variety of applications. MEMStechniques typically, but not in all circumstances, include thedeposition of thin films, etching using dry and/or wet methods, andlithography for pattern formation. Because MEMS is an offshoot of thesemiconductor industry, a vast worldwide manufacturing infrastructure isin place for cost-effective, high volume, precision production.Generally speaking, the more similar the full MEMS process is toexisting integrated circuit processes, e.g. CMOS compatible, the moreaccessible this infrastructure is.

The microstructure of the invention can be fabricated in many ways, suchas fabrication methods used for integrated circuits (e.g. interconnects)or MEMS. In the following, an exemplary fabrication method compatiblewith the MEMS fabrication for making a microparticle will be discussedwith reference to FIG. 5 and FIG. 6A to FIG. 6M, wherein themicroparticle comprises opaque segments that are composed of amorphoussilicon, and visible light transmissive gaps that are comprised ofsilicon dioxide. It will be appreciated by those skilled in the art thatthe following fabrication discussion is for demonstration purposes only,and should not be interpreted as a limitation on the scope of theinvention. In fact, many fabrication methods could be used withoutdeparting from the spirit of the invention.

Referring to FIG. 5, a silicon substrate is provided at step 122. Othersubstrates, such as glass wafers or glass panels could also be used (aswill be discussed further herein below). Assuming a silicon substrate,on the substrate is deposited a silicon dioxide layer at step 124. Thedeposition can be performed with many suitable thin film depositiontechniques, such as CVD, PVD, spin-on etc. as mentioned above. Anamorphous silicon layer is then deposited on the SiO₂ layer at step 126followed by deposition of a hard mask oxide layer at step 128. Thoughnot needed, the use of a hard mask reduces photoresist coating problemscause by topology, particularly when the amorphous silicon layer isrelatively thick (e.g. 1 μm or more in thickness). The hard mask oxidelayer is then patterned at step 130. With the patterned hard mask layer,the amorphous silicon layer is etched with a plasma etch so as to formthe desired pattern at step 132. A top SiO₂ layer is then deposited onthe patterned silicon layer at step 134 followed by patterning thesilicon dioxide layer at step 136 to form separate (but stillunreleased) microparticles. Then the microparticles are released fromthe silicon substrate at step 140 by a non-direction silicon etch thatetches into the silicon substrate and causes the microparticles to beseparated as individual particles. The flow chart in FIG. 9 as discussedabove can be better demonstrated in cross-sectional views and top viewsof the microparticle at different steps. The cross-sectional and topviews are schematically illustrated in FIG. 6 a to FIG. 6 m.

Referring to FIG. 6 a, SiO₂ layer 146, silicon layer 148, and hard masklayer 150 are sequentially deposited on silicon substrate 142. Hard masklayer 150 is then patterned so as to form segment strips (e.g. 152 and156) and gap strips (e.g. 154 and 158), as shown in FIG. 6 b. Thesegment and gap strips formed from the patterning of the hard mask layercorrespond to the segments and gaps of the target microparticle. Thesegment and gap strips are better illustrated in a top view of themicroparticle in FIG. 6 c. Referring to FIG. 6 c, segment strips (e.g.152 and 156) and gap strips (e.g. 154 and 158) are formed with layer 148that is visible from the top.

The patterning of the layers can be done in many methods, one of whichis photolithography that is widely used in standard fabrication forsemiconductor integrated circuits and MEMS devices. The most common formof photolithography used in the MEMS industry is contactphotolithography. A reticle (aka mask) is typically composed of a binarychrome pattern on a glass plate. The reticle is placed very near or incontact with a photoresist covered wafer (or other substrate). UV lightis shone through the mask, exposing the photoresist. The wafer is thendeveloped, removing the photoresist in the exposed regions (forpositive-tone photoresist). The pattern on the reticle is thustransferred to the photoresist where it serves as a mask for asubsequent etching step.

Projection photolithography is another type of photolithography that isused exclusively in modern integrated circuit manufacturing. Instead ofbringing the mask into physical contact, projection photolithographyuses a system of lenses to focus the mask pattern onto the wafer. Theprimary advantage of this system is the ability to shrink the maskpattern through the projection optics. A typical system has a five timesreduction factor. In general, much smaller feature sizes can be printedwith projection as compared to contact lithography. A projectionphotolithography system is also known as a step-and-repeat system (orstepper for short). The maximum pattern or field size on the mask issignificantly smaller than the wafer diameter. The mask pattern isrepeatedly exposed (“stepped”) on the wafer forming an array of “dies”.The stepping distance is the distance the wafer stage travels in X and Ybetween exposures and is usually equal to the die size. This typicalscheme produces a non-overlapping array of identical dies, allowing forsubsequent parallel processing of the dies on the wafer.

The hard mask layer (150) is further patterned so as to form discreteareas, as shown in FIG. 6 d and FIG. 6 e. As shown in FIG. 6 d, the hardmask layer 150 is patterned in the X and Y directions so as to formdiscrete hard mask areas (eg. areas 160, 162, 164, and 166 in FIG. 6 e).These discrete hard mask areas will in turn be used to form discretesilicon areas in the layer below.

In the example above, the patterning of the hard mask layer is performedin two separate lithography steps. In an alternative example, thereticle may comprise a pattern such that the patterning of the hard maskcan be accomplished with a single lithography step. As a furtheralternative, the hard mask can be omitted and either a two step orsingle step lithography process used.

After patterning the top hard mask layer, silicon layer 148 is etched soas to form corresponding discrete silicon areas on the substrate, suchas silicon segments 168 and 172, with areas there between for materialof greater transparency (e.g. gap areas 170 and 172, as shown in FIG. 6f). The top view of the microparticle as shown in FIG. 6 f isschematically illustrated in FIG. 6 g. As seen in FIG. 6 g, transmissivelayer 146 is now exposed when viewed from the top, with segments 160,162, 164, and 166 formed on transmissive layer 146. SEM images of thestructures at this point in the fabrication process are shown in FIG. 9Aand FIG. 9B. The structures have a very high degree of precision, e.g.vertical sidewalls and sharp corner. Of course more rounded structuresare also in the scope of these methods.

After patterning silicon layer 148, transmissive layer 168 is thendeposited as shown in FIG. 6 h. The more light transmissive layer 168may or may not be composed of the same material as the more lighttransmissive layer 146. A top view of the microparticles in FIG. 6 h isschematically illustrated in FIG. 6 i. A cross section view of amicroparticle is shown in FIG. 7. A perspective view of the particles onthe substrate is shown in FIG. 8.

The microparticles are then separated from each other, while stillattached to the underlying substrate, as shown in FIG. 6 j. FIG. 6 kschematically illustrates a top view of the microparticle in FIG. 6 j,wherein each microparticle is separated from adjacent microparticles,but surrounded by the light transmissive layer (i.e. layer 168 in FIG. 6h). Finally, the separated microparticles are detached from the siliconsubstrate 142, as shown in a cross-sectional view in FIG. 6 l. A topview of the detached microparticles from the silicon substrate isillustrated in FIG. 6 m. The detaching of the microparticles from theunderlying substrate (the “release” step) can be performed with anysuitable etchant—preferably a gas or liquid matched to etch in alldirections and undercut the microparticles. An additional sacrificiallayer can be provided on the substrate in place of etching into thesubstrate itself. The etching can be wet, dry, or plasma etching; andthe detaching layer is thus desired to be composed of a materialetchable with the selected etching method. In particular, the etchantcan be a spontaneous vapor phase chemical etchant such as aninterhalogen (e.g. BrF₃ or BrCl₃), a noble gas halide (e.g. XeF₂), or anacidic vapor such as HF. A liquid could also be used to release themicroparticles, such as TMAH, KOH (or other hydroxides such as NaOH,CeOH, RbOH, NH₄OH, etc.), EDP (ethylene diamine pyrocatechol), aminegallate, −HF etches glass so that won't work HNA (Hydrofluoricacid+Nitric acid+Acetic acid), or any other suitable silicon etchant(when the substrate or layer to be removed in the release is silicon(amorphous silicon or polysilicon or single crystal silicon—or tungsten,tungsten nitride, molybdenum, titanium or other material that can beremoved in a silicon etchant such as XeF₂). If the material to beremoved is not silicon, then the etchant is naturally matched to thesacrificial material (e.g. downstream oxygen plasma for a photoresist orpolyimide sacrificial layer, etc.).

The indentations are as a result of the particular fabrication method;and can remain in the final product, or can be removed by, for example,planarization—e.g. chemical-mechanical-polishing (CMP) techniques. Infact, the indentations in some situations can be beneficial for codedetection and/or fluorescence quantitation using fluorescent methodsbecause the binding of a fluorescently tagged material to the surface ofthe microbarcode is greater in the indentation areas (per unit length ofthe microbarcode), the so called indentation signal enhancement,fluorescence can be greater in the indentation areas and can be used todetermine the code (with or without other transmissive or reflectivetechniques discussed herein below). The same indentation signalenhancement would be applicable with reporter systems other thanfluorescence, e.g. radioactive reporters, etc.

Though a silicon wafer was mentioned as the substrate in the examplegiven above, a glass substrate, such as a glass wafer or larger glasssheet or panel (e.g. like those used in the flat panel display industry)could be used. Glass (or silicon) wafers can be of any suitablesize—e.g. 4 in., 6 in., 8 in. or 12 in. When a glass wafer is used,typically an additional sacrificial layer will first be deposited (forlater removal during the release step). The sacrificial layer can besemiconductor material, such as silicon, an early transition metal, suchas titanium, chromium, tungsten, molybdenum, etc. or a polymer, such asphotoresist, as mentioned earlier herein.

SEMs

A scanning-electron-microscopy (SEM) image of a segment (e.g. segment102) in FIG. 1 a is presented in FIG. 9 c. As can be seen in the figure,the cross-section of the segment is substantially square. The top of thesegment has a width of 1.0 micron; and the bottom width of the segmenthas a width of 1.2 microns. The height of the segment is approximately 1micron. Of course larger or smaller dimensions are possible.

An SEM image of a multiplicity of microparticles fabricated with theexemplary fabrication method as discussed above is presented in FIG.10A. The SEM image clearly illustrates the opaque segment 172 surroundedby transmissive material of the microparticle. Also, the indentationsmentioned previously are clearly visible. The sample in the SEM image ofFIG. 10A was prepared for characterization by cleaving a chipperpendicular to the long axis of the particles, followed by a timedsilicon etch to provide higher contrast between the inner silicon andouter silicon dioxide, purely for imaging purposes.

Release

The microparticles of the invention can be fabricated at thewafer-level, and released either at the wafer level or die level.Specifically, a plurality of dies each comprising a set ofmicroparticles can be formed on a wafer. The microparticles on each diemay or may not be the same—that is the microparticles on each die may ormay not have the same code. After forming the microparticles, the diescan be separated from the wafer; and the wafer(s) on the singulated diescan be then removed. An exemplary wafer-level fabrication method isdemonstrated in FIG. 13A to FIG. 13C.

Referring to FIG. 13A, a plurality of dies is formed on wafer 236. Inthis particular example, multiple microparticles are formed on each die.The number, such as 3, 221, or 967 on each die represents the codeincorporated in the microparticles in the die. The microparticles can beformed with a method as discussed above with reference to FIG. 6A toFIG. 6M. After formation of the microparticles but prior to release, thewafer can be partially cut, preferably to a depth about half the waferthickness. The wafer is then cleaned, for example with solvents and/or astrong acid (sulfuric, hydrogen peroxide combination). The clean is animportant step as it prepares a fresh glass surface for laterfunctionalization and biomolecule attachment. The clean can also beperformed after the wafer is separated into individual dies, or on theparticles once they have been released.

After the formation of the microparticles, the wafer is then broken intodies as shown in FIG. 13B, where each die preferably, but notnecessarily, contains a single code. The dies are then placed inseparate vessels such as test tubes or the wells of a well plate forrelease, shown in FIG. 13C. The well plate can be a typical 96-wellplate (or 24-well, 384-well, etc.), or any other suitable set of holdingareas or containers. For example, dies containing the numericallyrepresented codes: 3, 221, and 967, are placed in different tubes forrelease. By releasing, the microparticles are detached from the wafer;and the particles can fall into the solution in the releasing liquidwhen a wet etch is used. The microparticles over time settle to thebottom of the tube or well due to gravity (or the tubes can becentrifuged). In some applications, it may be desirable to releasemultiple dies comprising one or more codes into a single container.

FIG. 12A shows particles before release, and FIG. 12B shows the sameparticles (i.e. particles from the same die) after release. Both imagesare optical microscope images taken with a 100× air objective on anon-inverted inspection microscope. In FIG. 12B the particles are driedon a silicon chip.

The releasing step can be performed in many ways, such as dry etch, wetetch, and downstream plasma etch. In an exemplary bulk wet etch, shownschematically in FIG. 11A tetramethyl ammonium hydroxide (TMAH) is usedas the etching agent. TMAH can be heated to a temperature approximatelyfrom 70-80 C. Other chemical etchants can also be used and may workequally well, such as interhalogen (e.g. BrF₃ and ClF₃) and noble gashalide (e.g. XeF₂), HF in spontaneous vapor phase etch, potassiumhydroxide in a gas phase etch, KOH, and other suitable etchants. Ascreen having characteristic apertures (or filter membrane with pores)less than the smallest microparticle dimension can be placed on the topof each well or container, whether liquid or gas release is used, tokeep the codes safely within each container and avoid contamination ofmicroparticles into adjacent wells. During the etching, especially thegas phase etch or dry etch, a mesh can be attached to each tube, whetheron one end of the tube, well or container, or multiple mesh covering onmore than one side of a tube, well or container, such that gas etchantand etching products can flow freely through the mesh while themicroparticles are stopped by the mesh. A mesh or other filter can helpto drain the liquid release etchant as well, without releasing themicroparticles. Another example of a release etch process is shown inFIG. 11B and involves the deposition or formation of a sacrificiallayer, as has been previously described.

After pelleting the particles through centrifugation or lapse of time,the liquid (so called supernatant) is removed and the particles arewashed several times in water or a solvent. “Washing” refers to thesuccessive replacement of the supernatant with a new liquid, usually oneinvolved in the next chemical processing step. After detaching themicroparticles from the substrate (or wafer), the substrate can beremoved from etchant—leaving the microparticles in tubes. The releasedmicroparticles can then be transferred to containers for use.

The microparticles can be fabricated on the wafer level, as shown inFIG. 13 a to FIG. 13 c. Referring to FIG. 13 a, wafer 236, which is asubstrate as discussed above with reference to step 122 in FIG. 2,comprises a plurality of dies, such as dies 1 and 3. In an example ofthe invention, the wafer has 10 or more, 24 or more, 30 or more, or 50or more dies. Each die comprises a number of microparticles of theinvention, wherein the number can be 10000 or more, 20000 or more, or50000 or more. The microparticles in the same die are preferably thesame (though not required); and the microparticles in different dies arepreferably different (again, not required) so as to represent differentcodes. In the instance when different dies comprise microparticles ofdifferent codes, the dies are preferably assigned with uniqueidentification numbers, as shown in the figure so as to distinguish thedies and codes in dies.

Detection

FIG. 14 presents a reflectance-mode inverted microscope image of 8encoded microparticles of the present inventions. All such black andwhite microscope images with a black background are taken on an invertedepi-fluorescence microscope with the released particles in the well of awell plate. The particles are dispensed into the well in a liquid andsettle by gravity onto the bottom surface where they are imaged frombelow. Each particle in FIG. 14 has a different code. Segments of theless transparent material (e.g. opaque material in the visiblespectrum), in this case amorphous silicon, reflect light and are thebrighter regions in the image. The surrounding transparent material, inthis case silicon dioxide, is not visible in the reflectance-modeimages. The particles are 16 nm long by 2 nm wide and approximatelysquare in cross section. The image is a combination of selections from 8images, one for each code. The illumination light is at 436 nm, and theobjective used is a 60× magnification oil immersion lens.

FIG. 16 presents a full field, single image taken at the samemagnification as that in FIG. 14. The image is a mixture of manydifferent codes. All particles form a high density monolayer—that is,there is no particle aggregation or clumping. The characteristic of themonolayer formation is one of the key advantages of the microparticlesof the invention. When the microparticles are overlapped, aggregated, orclumped, the microparticles can not be properly identified. As aconsequence, microparticles that do not readily form monolayers asherein, are forced to be used at relatively low densities (the totalmicroparticles per unit area on the imaging surface). Low densityimaging translates to correspondingly low throughput for the number ofparticles measured per unit time. This low throughput can be alimitation in many applications

The tendency of the microparticles to form a monolayer is not trivial.Monolayer formation involves many factors, such as the surface chargestate (or zeta potential) of the microparticles, the density ofmicroparticles in a specific solution, the fluid in which microparticlesare contained, and the surface onto which the microparticles aredisposed. Accordingly, the microparticles of the invention are comprisedof materials and are constructed in a form that favors the maintenanceof a charged state sufficient to substantially overcome stiction forces;and thus microparticles are capable of undergoing Brownian motion whichfacilitates the formation of a reasonably dense monolayer of particles.

In biological applications, the microparticles are often used to carrybiochemical probe molecules. For immobilizing such probe molecules, themicrostructure preferably comprises a surface layer, such as a silicondioxide layer, which can be chemically modified to attach to the probemolecules. In accordance with an example of the invention, themicroparticles are constructed such that the microparticles are capableof forming a monolayer, for example, at the bottom of a well containinga liquid; and the monolayer comprises 500 or more particles per squaremillimeter, more preferably 1,000 or more, 2,000 or more, or 3,000 ormore microparticles per square millimeter. In an alternative example,the microparticles can form a monolayer that such that the detectableparticles occupy 30% or more, 50% or more, or 70% or more of the totalimage area (i.e. the image field of view). In connection with theexample mechanism of self-assembled monolayer formation, it is preferredthat the 2D diffusion coefficient of the microparticles of the inventionis greater than 1×10⁻¹² cm²/s. For accommodating the monolayer of themicroparticles, the container for holding the microparticles indetection preferably has a substantially flat bottom portion.

FIG. 15 shows a diagram of an optical system used to image the encodedmicroparticles of the invention. The optical system 254 can be used toread the microparticle codes, including for bioassay applications. Thesystem is an inverted epi-fluorescence microscope configuration. Otherexemplary optical microscopy systems for the detection of themicroparticles of the invention include but are not limited to confocalmicroscope systems, Total Internal Reflection Fluorescent (TIRF), etc.Well plate 257 contains many wells of which a single well 256 is imaged.The well plate sits on microscope stage 258. Microparticles that havebeen dispensed into well 256 in a liquid settle by gravity to the bottomsurface. Light coming from light source 268 passes through excitationfilter 266 which selects the illuminating wavelength. The illuminatinglight reflects off beam-splitter 262 and travels up through objective260. Typically, only a fraction of well 256 bottom surface area isimaged. The imaged area is referred to as the “field” or “field area”.Reflected or emitted light (know together as collection light) travelsback down the objective and passes through the beam-splitter 262.Emission filter 270 selects for the collection wavelength. Finally thecollected light is recorded with a detector 272, such as a CCD camera.This simplified version of the optical system is not meant to becomplete. In practice, the actual microscope may have many morefeatures, preferably including an automated stage and auto focus systemfor high throughput imaging. The excitation filter and emission filtercan be mounted on computer controlled filter wheels and areautomatically changed for the reflectance and fluorescence images. Acomputer controlled shutter controls the exposure times.

FIG. 43 shows a diagram of an optical system used to image encodedmicroparticles that utilizes two CCD cameras for the simultaneousacquisition of a reflectance and fluorescence image.

The optical system is used for detection in bioassays. The system is aninverted epi-fluorescence microscope configuration. In the preferredembodiment, a wellplate 201 contains many wells of which a single well203 is imaged. The wellplate 201 sits on the microscope stage 209.Particles that have been dispensed into the well 203 in a fluid settleby gravity to the bottom surface. Light coming from the light source 215goes through the excitation filter 219 which selects the illuminatingwavelength. The illuminating light reflects off the beam splitter 213and travels up through the objective 211. Typically, only a fraction ofthe well 203 bottom surface area is imaged. The imaged area is referredto as the “field” or “field area”. Reflected or emitted light (knowtogether as the collection light) travels back down the objective andpasses through the first beam splitter 213. The collection light thenpasses through the second beam splitter 217 which breaks it into thereflectance path and the fluorescence path. The emission filter 221 islocated in the fluorescence path and selects for the appropriatefluorescence emission wavelength. The light in the fluorescence path isrecorded with the fluorescence CCD camera 223. The light in thereflectance path is recorded with the reflectance CCD camera 225. Thissimplified version of the optical system is not meant to be complete. Inpractice, the actual microscope system may have more features,preferably including an automated stage and auto focus system for highthroughput imaging. The excitation filter 219 and emission filter 221may be mounted on computer controlled filter wheels to be automaticallychanged for multi-fluorophore experiments. A computer controlled shuttermay be used to control the exposure times.

The system depicted in FIG. 43 is an improvement over the standard onecamera system that utilizes filter wheels (or filter cube wheels) toacquire reflectance and fluorescence images in succession. The inventionis accomplished by splitting the outgoing beam path into two componentswith a beam splitter. One component is the reflectance path, which iscaptured with one CCD camera. The other component is the fluorescencepath, which is filtered for the appropriate wavelength and captured witha second matched CCD camera. The beam splitter can be designed such thatmore light is directed into the fluorescence path such that the exposuretimes on the two cameras are approximately equal. The two camera systeminvention offers the advantage of increased throughput. Additionally,the invention offers the advantage of eliminating the positional shiftsbetween reflectance and fluorescence images pairs that may be present inthose of the one camera system. This simplifies the computer softwarebased processing of image pairs because the particles are in the samephysical locations in both images of the image pair. In a furtherembodiment, the optical system is used for detection in bioassays.

FIG. 17 shows a high magnification image of encoded microparticles. Theimaged particles consist of discrete segments of varying sizes. Thesmallest size segments 20 are 0.6 μm. End segments 22 form the end of asingle particle. An example of the invention consists of encodedmicroparticles with spatial encoding features less than 1.5 μm in size.

FIG. 18 a shows a montage of 12 dense reflectance images of encodedmicroparticles. Approximately 6,000 particles are in the images. Theparticles are a small fraction of the approximately 200,000 particlestotal in a well of a 384 wellplate. The total particles areapproximately 10% of a set that contains 1035 codes (batches). The setwas formed by combining approximately 2,000 particles from each of the1035 batches where each batch contained approximately 2 millionparticles of a single code. These images are a subset of a larger imageset from which data regarding identification accuracy is presentedbelow.

FIG. 18 b shows a transmission fluorescence microscope image of examplemicroparticles of the invention. Shown are here, in addition, small,elongated, encoded microparticles with an outer surface that is entirelyglass. Shown are a multiplicity of non-spherical encoded particles witha silica (e.g. glass or silicon dioxide) outer surface and a length lessthan 70 μm (e.g. less than 50 μm). The length of the example particlesin this particular example is 15 μm.

In this image, the particles are in a solution that contains suspendedfluorescent molecules. The fluorescent molecules, when excited by themicroscope light source, provide illumination from above (i.e. behindwith respect to the collection optics, see FIG. 15 for a diagram of thebasic optical system) the particles. This image is similar to one thatwould be provided in transmission mode imaging configuration, and unlikethe reflectance mode images of FIG. 16 to FIG. 18A, clearly shown theouter glass surface of the particles.

For successfully identifying the microparticles, e.g. reading the codesincorporated therein, the images of the microparticles may be processed.Such image processing can be performed with the aid of softwareprograms. According to examples of software programs and algorithms,pairs of raw and processed image are presented in FIG. 19A and FIG. 19Band in FIGS. 20A and 20B.

FIG. 19A shows a full field reflectance image; and FIG. 19B shows thesame image selection of FIG. 19A after the image processing to associatediscrete segments into full microparticles. The particles shown in theimages are of a single code. Images of encoded microparticles of thepresent invention consist of discrete segments that appear white in thereflectance imaging. The gaps, which are between segments of individualmicroparticles consist of glass, are transparent, and therefore appearblack in the reflectance image. The background of the images is alsoblack. The segments are associated together into the particles by analgorithm. The algorithm finds the long axis of a long segment andsearches along that axis for segments. Segments are accepted or rejectedbased on predefined parameters. The black lines in FIG. 19B correspondto particles for which segments have been associated together. In anexample of the aforementioned algorithm, a computer program product thatidentifies the codes of encoded particles by associating discreteregions in an image into individual particles.

FIG. 20A shows a selection of a reflectance image; and FIG. 20B showsthe same image selection of FIG. 20A after the image processing toassociate discrete segments into full microparticles. The particlesshown in the images are of a multiplicity of codes. The segments of theparticles are numbered. The black lines in FIG. 20B are drawn toillustrate the segments that have been grouped together into particlesby the image processing software.

Referring to FIG. 21, a processed image is shown on the right and pixelintensity profiles from 4 example microparticles are shown on the left.The pixel intensity profiles are further processed by a computersoftware program to determine the codes of the microparticles. Byidentifying the center locations of the gaps, as indicated by circles inthe pixel intensity profile in the lower left, the codes of themicroparticles can be identified. As mentioned previously, the centergap locations are not sensitive to variations in both the particlefabrication process or image processing, i.e. variations in thedimensions of the actual segments and gaps that make up the examplestructure of FIG. 1A. This feature is highly advantageous as it providesrobust and accurate code identification of the encoded microparticles.

Table 3 shows identification data for image sets that include thoseimages shown in FIG. 18A.

TABLE 3 Full ID % Limited ID % Images 30,069 Codes 1,035 Codes 40xobjective  99.5%  99.98% ~500 particles/image 9866 particles measured60x objective 99.85% 99.995% ~250 particles/image 2733 particlesmeasured

The microparticles included in Table 3 have a codespace of 30,069,wherein the codespace is defined as the total number of possible codeswith the particular particle design, i.e. with the chosen coding schemeand coding scheme parameters. A pre-determined identification methodassigns one of the 30,069 possible codes based on the analysis of theparticle segment information. 1035 codes were randomly selected,manufactured, and mixed to form the collection. When analyzing theidentification of the collection, if the software assigned code is oneof the 1035, it is assumed to be correct. The number of “correctly”identified particles divided by the total is called the “ID %”. Thisassumption underestimates the error rate (1−ID %) by the probabilitythat a random error falls within the 1035 present codes, or 1035 dividedby 30,069=about 3%. The assumption therefore ignores this 3% deviationand provides a close approximation to the true identification accuracy.

FIG. 22 shows a schematic of a specially prepared surface that havefeatures designed to immobilize and separate the encoded microparticlesfor imaging. The surface includes features, e.g. grooves and/or pitsthat trap the particles. Such surfaces could be useful in applicationswhere the particles experience increased aggregation due to the natureof molecules coated on the surface or properties of the imaging medium.FIG. 22 shows an example of such a substrate 320 with grooves 322designed to capture the particles. The substrate 320 is preferred to beglass, but may be other materials, for example other transparentmaterials. The grooves 322 shown in FIG. 22 have a V-shape but may takeon any shape such as having a square or U-shaped bottom thataccomplishes the task of capturing the particles. When particles areplaced onto the surface, particles 324 fall into the grooves and areimmobilized. In an example, encoded microparticles of the presentinvention, having an elongated and substantially square cross section,may be immobilized in grooves having a flat bottom.

In an alternate example, a flow-cell enabling the microparticles flowingin a fluid can be provided for detection by continuous imaging, as shownin FIG. 23 and FIG. 24. Referring to FIG. 23, reflectance andfluorescence image pairs are acquired with the optical system depictedin FIG. 6 while the well plate is replaced with flowcell 320. Encodedmicroparticles 322 flow in a carrier fluid. Flow may be driven bypressure (hydrodynamic) or electrical means (electro-phoretic orelectro-osmotic). Further, microparticles may be aligned with electricor magnetic fields. The flow is from the left to the right as indicatedby the arrow. The upper figure of FIG. 23 shows the flow cell at a giventime and the lower figure of FIG. 23 shows the same flow cell at asubsequent time such that the particles have displaced a distance equalto approximately the length of the field of view. The optical systemobjective 330 is shown below the flow cell but may also be placed abovethe flow cell. In addition, the flow cell can be placed in otherconfigurations with, for example, the flow being directed vertically.The objective 330 images the capture field area 328. The first fieldarea 324 and the second field area 326 are shown as shaded regions. Inthe upper figure the first field area 324 overlaps with the capturefield area 328 and therefore the first field area 324 is imaged. In thelower figure the second field area 326 overlaps with the capture fieldarea 328 and therefore the second field area 326 is imaged. Byappropriately matching the flow speed, flow cell size, and opticalsystem, all particles passing through the flow cell can be imaged,thereby providing a system for high throughput detection. Anotherexemplary system for high throughput flow based detection of the encodedmicroparticles of the invention is a flow cytometer, the methods andapplications thereof are well known in the art.

As depicted in FIGS. 14 and 19-21, it can readily be appreciated thatthe images of microparticles may easily be scanned and digitized usingCCD or CMOS type cameras and related technology. Once this digitalinformation is processed and stored in a computer on computer readablemedium. Various software may be employed to display the images and toidentify the codes, etc. The codes may the be correlated with whateverprobe or molecule has been attached to the encoded microparticle havingthat specific code using a look up table, also stored on the computerreadable medium.

Software programs have also been developed which can use these digitaldata and visual processes to distinguish between the particles. That is,once the particles are used in an assay, some will be labeled with, forinstance, one or more fluorophores, while others which did not bindtheir target will not be labeled. A software program may be used whichis able to distinguish between these two possibilities. However, itbecomes more problematic when the particles are close in proximity onthe bottom of the well plate and the fluorescence or other signal fromone encoded microparticle “bleeds” over in the detection apparatus andmakes it seem like unlabeled encoded microparticles are actuallylabeled, i.e. producing false positive results.

False positives may be removed by use of software programs that examinethe intensity of the label signal emanating from the encodedmicroparticle which is labeled. This concept is depicted in FIG. 53. Thethree boundaries drawn around the encoded microparticle may be anydistance from the microparticle that is optimized and convenient for thelabel and assay employed. The software determines the strength of thesignal from the label within each of the boundaries as a sum total.Depending on how rapidly the signal intensity drops off in each of thefurther-removed fields, the encoded microparticle may be identified as afalse positive. In other words, if a first field defined by theboundaries of the encoded microparticle itself appears to be emanating asignal, the software then looks at the next boundary (depicted as adashed line in the two-dimensional drawing of FIG. 53) and determinesthe intensity of the signal within that boundary. If the intensity dropsoff precipitously as compared to a positive standard control, then it ismore likely than not this signal is indicative of a false positive.However, if the intensity of the signal within each of the definedboundaries around the encoded microparticle matches that of a positivecontrol, then it is more likely than not that the encoded microparticleis not a false positive.

The false positive identification software is controlled by an algorithmwhich first identifies each microparticle at the bottom of the wellplate, then defines various theoretical 2-dimensional boundaries in theplane of the microparticle. The algorithm then determines the signalintensity within each of the boundaries and compares these values tothat of various positive controls. The algorithm can then eliminatevarious microparticles that are false positives depending on how thesignals within the variously assigned boundaries compare with thepositive controls.

This algorithm and software technique for determining false positives isespecially useful for encoded microparticles that tend to “bunch up” orfall to the bottom of the well plate in clumps such that multipleencoded microparticles, some labeled and some not, are overlapping eachother.

Thus, optical apertures may be defined by the user, which areprogressively larger in 2-dimensions than the size of the microparticle(see fields marked 1, 2 and 3 in FIG. 53). The measured label signal foreach increasingly larger field of detection around the microparticle maythen be compared.

If the detected label signal increases as the field is enlarged, i.e.more signal is detected in Field 3 than in Field 1, the microparticlemay be identified as a false positive, i.e. no label actually present onthat specific encoded microparticle in the experiment.

If the detected label signal decreases as the field is enlarged, i.e.less signal in Field 3 than in Field 1, the microparticle may beidentified as a true positive signal in the experiment.

Other Structures

Another alternative microparticle of the invention is schematicallyillustrated in FIG. 25. Referring to FIG. 25, microparticle 274comprises opaque segments, such as 276, and gaps, such as 278, which aretransmissive to the visible or near-visible light. The opaque materialcan be composed entirely or partially of a magnetic material such as(but not limited to) nickel, cobalt, or iron. The magnetic materialcould be incorporated as a thin layer 280 sandwiched between anothermaterial that forms the majority of the opaque material. The magneticmaterial gives the particles magnetic properties such that they can bemanipulated by magnetic fields. This can aid in particle handling orfacilitate the separation of biomolecules.

FIG. 26 shows a diagram of a spatially optically encoded microparticlewith a fluorescent outer layer 406. This invention has utility in thetagging of material goods whereby the fluorescent layer improves theability to easily find and identify the particles against diversebackgrounds. In an example, the fluorescent outer layer 406 is grownusing a modified version of the Stöber process [Van Blaadern, A.; Vrij,A.; Langmuir. 1992. Vol. 8, No. 12, 2921]. The fluorescent outer layer406 makes the entire particle fluorescent and facilitates the finding ofthe particles during detection. The reading of the particle code can beaccomplished by imaging the particle in reflectance or fluorescencemode. One may be preferred over the other depending on the application,medium in which or surface to which the particles are applied. Particlesof a single code can be used or mixtures of particles of different codescan be used. The particles can be applied in a medium such as a lacquer,varnish, or ink. The particles may be used to tag paper or fibers. Theparticles may be used to tag objects made of metal, wood, plastic, glassor any other material.

In another example, the fluorescent layer may be comprised offluorophores, or other luminescent materials. The fluorescent layer mayinteract with molecular species in an assay, for example withfluorescently labeled nucleic acids or protein samples via FluorescenceResonant Energy Transfer processes. In yet another example, themicroparticles may have a non-fluorescent layer, wherein incorporated inor on the layer are molecules, for example quenchers that interact withluminescent emitter molecules.

FIGS. 27 a to 27 c show schematic diagrams of encoded microparticles ofthe present invention with surface indentations that form a spatialcode. The microparticle may be fabricated by many methods including theaforementioned examples. FIG. 27 a has surface indentations, aka divots,e.g. grooves, only on the of face of the structure. FIG. 27 b has divotson two faces. In other examples, divots and other desirable surfacefeatures may be placed on one or more surfaces of the microparticlestructures, so as to provide a spatial code. FIG. 27 c shows anotherexample of such a structure, whereby the overall shape of themicroparticle is substantially cylindrical. In an example method ofmaking the microparticle of FIG. 27 c, optical fibers having a diameterless than 1 mm may be laser or tip scribed to form the indentations. Thecomposition of the structures of FIGS. 27 a to 27 c may be selected froma wide variety of materials, with glass being a preferred example.

In examples of encoded microparticles comprising indentations, thesurface of the particles have fluorescent, or otherwise emitting,molecules attached to or in the surface, as shown in FIG. 27 d. Theemitting molecules may be covalently attached to the surface, adsorbedto the surface, or otherwise bound to the surface. In an example, theemitting molecules are incorporated into a layer which is deposited ontothe microparticle. A uniform surface coverage of emitting molecules,e.g. a constant number of fluorophores per unit area, results in anonuniform aerial density. Aerial density is defined as an intensity perunit length or per unit area that is integrated through a depth of fieldin an optical image plane. In this example, the aerial density ismeasured as a signal intensity profile measured by a detector, forexample a CCD camera or photomultipler tube. FIGS. 28 a to 28 c show thenonuniform aerial density measured normal (i.e. perpendicular) to theparticle surface for corresponding particles in FIGS. 27 a to 27 c. Thesignal intensity profile has peaks corresponding to the location of thesurface indentions of the particles, which thus provide a detectable anduseful code. The surface features of the encoded microparticles of FIGS.27 a to 27 c may be detected by methods other than the use of emittingmolecules, including but not limited to the measurement of lightscattering, e.g. darkfield optical microscopy, etc.

As already mentioned, the encoded microparticles may be produced toinclude magnetic, ferromagnetic, diamagnetic, paramagnetic andsuperparamagnetic properties. To achieve this property, a thin layer offerromagnetic film may be patterned by standard semiconductor processes.However, sometimes these standard processes can yield magnetic encodedmicroparticles that retain hysteresis and remnant magnetism due to theintrinsic ferromagnetic properties of the material. These properties cansometimes, in certain solutions and buffers, especially those with ahigh salt concentration, cause the encoded microparticles to form clumpsand/or to behave erratically when placed inside an electromagneticfield, such as field present in standard electronic microscopes,scanners and CCD cameras. To produce encoded microparticles that do notpossess a magnetic moment in the absence of applied field, theferromagnetic film may be optionally substituted with material that isparamagnetic in nature.

In this process, the magnetic film may be substituted with aspin-on-glass (SOG) layer deposited on top of a reflective polysiliconlayer. Embedded within the spin-on-glass may be, for instance,nanometer-sized ferromagnetic beads or other particles (can bemanufactured or commercially available from companies such as, forinstance, Ocean Nanotech, LLC, Springdale, Ark.; TurboBeads, LLC,Zurich). By themselves, ferromagnetic nanoparticles behave assingle-domain magnets with a fixed moment, but when placed in a largegroup present in a monolayer within the present encoded microparticles,the thermokinetic energy of each fixed moment of each nanoparticle isenough to randomly orient the moments so that in the absence of appliedelectric field the matrix essentially remains net neutral in magnetism.This solution then effectively eliminates any potential clumping thatmay occur in certain conditions when using magnetic encodedmicroparticles in biological assays.

The generation of a matrix containing single-domain ferromagneticmaterial to create a super-paramagnetic material has been reported.(See, Brunsman et al., “Magnetic properties of carbon-coated,ferromagnetic nanoparticles produced by a carbon-arc method,” J. Appl.Phys., 75(10):5882-5884, 1994; Ramprasad et al., “Magnetic properties ofmetallic ferromagnetic nanoparticle composites,” J. App. Phys.,96(1):519-529, 2004; Liu, J. P., “Ferromagnetic nanoparticles:synthesis, processing and characterization,” JOM Journal of theMinerals, Metals and Materials Society, 62(4):56-61, 2010; and Petracicet al., “Collective states of interacting ferromagnetic nanoparticles,”J. Magnetism Magnetic Materials, 300(1):192-197, 2005, all of which areincorporated herein by reference in their entireties for all purposes).However, the application of this concept into a spin-on-glass materialthat is then used in the creation of the present encoded microparticlesprovides a unique solution to those who wish to use magneticmicroparticles for the purpose of providing an efficient and easy meansof isolating the encoded microparticles, for instance during wash stepsin the biological assay, etc. Furthermore, since the fabrication stepprimarily involves the etching of oxides, the technique of using aspin-on glass, itself an oxide polymer, lends itself to easy integrationinto the manufacturing processes described herein.

Method for Producing Codes

The invented general method of generating the codes on microparticlesconsists of the use of multiple lithographic printing steps of a singlecode element per particle region. The multiple printing steps createmultiple code elements per particle region. The code elements takentogether form the code for the microparticle. In a preferred example,the printing steps are performed on many particles in parallel using amaster pattern. A master pattern comprises an array of single codeelements per particle region. A code element may represent more than onephysical feature, such as holes, stripes, or gaps. The master pattern isprinted multiple times such that a multiplicity of microparticles withcomplete codes is formed, wherein the multiplicity of microparticlescomprises identical particles (e.g. all particles have the same code).Variations upon this theme, for example wherein the multiplicity ofmicroparticles are not identical, are anticipated and will be describedin detail below. Between multiple print steps, a component of theoverall printing system changes to translate the code element within theparticle region. In a most preferred example, this change is a movementof the substrate on which the particles are formed. In another preferredexample, this change is the movement of the master pattern. In yet otherexamples this change is the movement of an optical element such as amirror.

An example of the general method of generating code using multiple printsteps involves photolithography as the printing mechanism, e.g. contactphotolithography and projection photolithography. An example ofprojection photolithographic utilizes a step and repeat system (akastepper). A reticle contains a code pattern that has a single codeelement per particle. Through multiple exposures of this code pattern atdifferent lateral offsets, a multiplicity of code elements (perparticle) is created. Combined, these code elements form a completecode. The lateral offsets define the code and are programmed into thestepper software. The offsets, and therefore the code, can be changed ona per die or per wafer basis. The codes printed on different dies on awafer and/or different wafers in a lot are thus controlled by softwareand can be arbitrarily changed. This enables a powerful flexibility inthe manufacture of large sets of codes. A single mask set, having one toa few masks, can be used to generate an arbitrary number of codes,numbering into the 10⁵ range and beyond.

FIG. 29A to FIG. 29C shows an example of the invented method ofproducing the codes for microparticles. The microparticle regions 290are areas that, upon completion of the fabrication process, will bediscrete particles. FIG. 29A shows the status after the printing of thefirst code element 292 in each microparticle region 290, in this examplethe code elements are vertical stripes. FIG. 29B shows the status afterthe printing of a successive code element 294 in each microparticleregion 290. FIG. 29C shows the status of the printing of three more codeelements 296 in each microparticle region 290. The multiple printingsteps thus provide codes on the microparticles.

FIG. 30A to FIG. 30C shows another example of the invented method ofproducing the codes for microparticles. The microparticle regions 300are areas that, upon completion of the fabrication process, will bediscrete particles. FIG. 30A shows the status after the printing of thefirst code element 302 in each microparticle region 300, in this examplethe code elements are circular. FIG. 30B shows the status after theprinting of a successive code element 304 in each microparticle region.FIG. 30C shows the status of the printing of three more code elements306 in each microparticle region 300. The multiple printing steps thusprovide codes on the microparticles.

FIG. 31A to 31C show drawings of the 3 mask fields of the preferredembodiment of the microparticle structure and FIG. 31D shows a drawingof a reticle plate. FIG. 31A to 31C are small representative areas ofthe much larger full field (only 46 of approximately 2 million particlesare shown). In these drawings, the regions that are gray have chrome onthe actual reticle (so called “dark” in reticle terminology), and theregions that are white have no chrome (so called “clear”). Physically,the reticles are glass plates that usually measure 5″ to 6.25″ squareand are about 0.09″ thick. They are coated with a thin (a couple hundrednm) layer of chrome. The chrome is patterned with a resist through aserial lithography process, usually using a laser or ebeam system. Thereticle is then wet etched which selectively removes the chrome. Thefinal reticle then consists of a glass plate with chrome on one side inthe desired pattern.

The code pattern, shown in FIG. 31A, has vertical stripes 110 that areclear. There is one vertical stripe per particle. FIG. 31B shows the barpattern, which consists of horizontal stripes 112 that are dark (orequivalently wider horizontal stripes that are clear). The outlinepattern, shown in FIG. 31C, consists of rectangles 114 that are dark.Clear streets 116 extend in the horizontal and vertical directions,separating the rectangles 114. The rectangles 116 will form the outerborder of the particles. The horizontal stripes 112 define the width ofthe inner segments of opaque material. The vertical stripes 110 form thegaps in the segments. The gaps both form the code in the particle andseparate two adjacent particles. FIG. 16D shows a full reticle plate.The reticle field 118 is the center region of the reticle which containsthe pattern to be exposed. Alternate examples of the patterns describedare also envisioned, including combining the code and bar pattern into asingle pattern that can used according to the described multi printmethod.

An example of the invented method for producing codes usesphotolithography and positive-tone photoresist. Positive-tone means thatthe areas exposed to light are developed away. For a negative-toneresist, exposed regions are what remain after development. Thephotocurable epoxy SU-8 is an example of a negative-tone resist. In analternate example using a negative-tone resist such as SU-8, the regionsthat are to be segments are exposed to light instead of the regions thatare to be gaps.

FIGS. 52A to 52C show flowcharts of examples of the code elementpatterning and etch steps. FIG. 52A shows the case where a hard mask isnot used. This process is simpler but may produce segments with roundedcorners because of the proximity effect of the photoresist exposures. Atthe corners of the segments, the photoresist gets some residual exposurefrom both the vertical stripes of the code pattern and the horizontalstripes of the bar pattern. The resulting rounding of the corners,though within the scope of the invention, is less desirable because itproduces final particles that look different from the side vs. the topand bottom surfaces. The extent to which the rounding occurs depends onthe specifics of the photolithography process including the pattern onthe reticles, wavelength of the light source, and photoresist. FIG. 52Bshows an example of the multi print method based patterning process andis described in detail in the below FIGS. 33A to 33M and FIGS. 34A to34M. FIG. 52C shows another example of the particle fabrication processwhere instead of transferring the bar pattern to the hard mask, the barpattern photoresist is used as the mask in conjunction with the hardmask oxide. This example method eliminates a few steps but may not beappropriate depending upon the specifics of the poly etch chemistry.

An alternate example of the general method of generating code usingmultiple print steps utilizes stamping (aka imprint lithography) as theprinting mechanism, and is schematically depicted in FIG. 32. FIG. 32schematically shows a small region of an example master pattern forstamp printing according to the invented multi-print-steps-to-build-thecode-up method, e.g. 1) stamping or pressing a stamper apparatus intothe particle containing substrate, followed by 2) moving either thestamper apparatus or the substrate, and 3) stamping at least one moretime in a nearby location, such that a complete code on themicroparticles is formed. The substrate on which the microparticles canbe formed using imprint lithography may be a wafer, such as a 100 mm,150 mm, 200 mm, or 300 mm silicon wafer, or a panel, such as a 5″ orlarger glass or quartz panel, or rolled sheets (including but notlimited to polymeric sheets).

FIGS. 33A to 33M and 34A to 34M illustrate the microfabrication processsteps of the example encoded microparticle of FIG. 1A. These stepsdefine the inner opaque segments (which contain the code). The steps areshown in more detail than in FIG. 6 a to FIG. 6 m and include thephotoresist exposure and development. FIG. 33 a to FIG. 33 m show topdown drawings and FIG. 34 a to FIG. 34 m show the corresponding crosssectional views. The cross-section line 50 is shown in FIG. 33A to FIG.33M. In FIG. 33A, the top surface is the hard mask oxide 58. In FIG.34A, the film stack on the starting substrate 52 consists of the bottomoxide 54, poly 56, and hard mask oxide 58. In FIG. 33B the wafer hasbeen coated with unexposed photoresist 120. The unexposed photoresist120 is shown as the top layer in FIG. 34B. In FIGS. 33C and 34C, theunexposed photoresist 120 has been exposed with the code pattern asingle time, forming exposed photoresist 122 regions. In FIGS. 33D and34D, the code pattern has been exposed multiple times with lateraloffsets applied between the exposures. In the preferred embodiment, thecode pattern is exposed twice in directly adjacent regions to formdouble width stripes 124. Single width stripes 126 are the “gaps” thatform the code. The double width stripes 124 are located in between theparticles and separate the particles. To clarify, the lateral offsetsare achieved by moving the stage on which the wafer sits. The lateraloffsets are programmed into the stepper software. The lateral offsetsdefine the code of the microparticles on that die. The lateral offsets(and thus code) can be different for every die on a wafer. Each wafer ina lot of wafers can have a different set of codes. In this way, verylarge code sets can be realized.

FIGS. 33E and 34E show the wafer after development of the photoresist.The exposed photoresist 122 from FIGS. 33D and 34D is removed revealingthe underlying hard mask oxide 58. FIGS. 33F and 34F show the waferafter the oxide etch. The oxide etch removes the hard mask oxide 58 inthe exposed regions revealing the underlying poly 56. FIGS. 33G and 34Gshow the wafer after the unexposed photoresist 120 of FIGS. 33F and 34Fis removed. The hard mask oxide 58 is present in the regions that willbecome the segments. The poly 56 is exposed in the regions that willbecome the gaps in the opaque material. FIGS. 33H and 34H show the waferafter it is again coated with unexposed photoresist 120.

FIGS. 33I and 34I show the wafer after the exposure of the bar pattern.This is just a single exposure and is the same on all dies. Thisexposure is preferably aligned to the pattern already on the wafer.After exposure, the unexposed photoresist 120 pattern consists ofhorizontal stripes which define the segment width. The exposedphotoresist 122 pattern consists of horizontal stripes which define thehorizontal separations between the segments. FIGS. 33J and 34J show thewafer after the development of the photoresist. The exposed photoresist122 from FIGS. 33I and 34I is removed revealing the underlying hard maskoxide 58 and poly 56. FIGS. 33K and 34K show the wafer after the oxideetch of the hard mask oxide. Only the poly 56 is present in the exposedphotoresist region of FIG. 33I. FIGS. 33L and 34L show the wafer afterthe unexposed photoresist 120 is removed. At this point in the process,the top surface of the wafer is poly 56 with hard mask oxide 58 coveringthe poly 56 in the regions which are to become the segments of opaquematerial. Finally, FIGS. 33M and 34M show the wafer after the poly etch.The poly etch removes the poly 56 of FIGS. 33L and 34L, revealing theunderlying bottom oxide 54. The hard mask oxide 58 is still present onthe top surface of the poly 56 in the segment pattern.

In addition to the microparticle as illustrated in FIG. 1A, the methodsabove can be used to produce the codes for other encoded microparticledesigns including currently known particle designs as well as otheralternative designs. The method above can be used to produce the codesfor the encoded microparticles, for example, in FIGS. 35A to 35C.

Referring to FIG. 35A, a bar-shaped microparticle with code elementsconsisting of holes such as holes 178 and 180 that are surrounded byframe material 182. The number and the arrangement of the holes forms acode derived from a predetermined coding scheme.

FIG. 35B shows another bar-shaped particle with the code elementscomprising notches, such as notch 196. The adjacent notches define a setof protruding structures with different widths. The total number ofprotruding structures and the arrangement of the protruding structureswith different widths represent a code derived from a coding scheme.FIG. 35C shows a square plate shaped particle with the code elementsconsisting of holes, such as holes 200 and 202 that are separated by gap202. The plate particle also includes an indentation 198 in one cornerto break the symmetry of the particle and thus allow for more codes.Further shapes and code element architectures can also be made with theaforementioned method of producing codes.

FIG. 36 shows four microscope images of actual encoded microparticles,just prior to release from the dies. These particles are producedaccording to the invented technique of producing codes with multipleprint steps and according to designs described above.

FIG. 37 shows charts of example data that is input into the steppersoftware to generate different codes on every die on a wafer. The chartsshow which dies get printed in 9 different passes and with what offsets.The data shown in FIG. 37 is an example of one system for organizing themulti print method using a stepper for providing a multiplicity of codeson a multiplicity of dies on a wafer. In this example, each die isexposed at most one time during a single pass. A wafer map of which diesare to receive exposures during the stepper exposure passes in thisexample is shown in the column on the left. “1” designates exposure. “0”designates no exposure. The middle column shows a wafer shot map of theexposure offsets, designated with offset letters “A”, “B”, “C”, and “D”.The right column shows a lookup chart of 1) the exposure locationrelative to the end of the particle, 2) the offset letter, and 3) theexposure locations programmed relative to a stepper reference point. Therows correspond to the different passes, 9 in this example.

Another example of a system for organizing the multi print method usinga stepper is to exposure all of the code elements within a single diebefore moving on to the next die. Of course, a number of offsets otherthan four could be used. Though this and other examples of the generalmethod of producing codes on microparticles has been described withrespect to using a projection photolithography and a stepper, contactlithography and other patterning methods may also be used.

FIG. 38 shows drawings of an example scheme for producing an increasednumber of codes per die. In this scheme, within a die there are fixedand variable code element locations. Dies are divided into sub regionswhere each sub region has a different pattern of fixed code elements.For each die, a different pattern of variable code elements is exposed.The fixed and variable code elements together make up the entire code. Asingle wafer thus contains a total number of codes equal to the productof the number of dies per wafer and sub regions per die. An individualdie, containing sub regions of different codes, could be physicallyseparated into smaller sub-dies and the different codes released intodifferent tubes. An alternative is to keep the dies intact and releasethe whole die into a single tube. This would create a mixture of codesfrom the different sub regions. This approach may be particularly usefulfor combinatorial synthesis applications.

The invented method of producing codes, for example the use of aphotolithographic step and repeat system to form a complete code throughmultiple exposure steps of a single reticle field, may be used to applyunique codes to many types of components, e.g. MEMS and IC devices.

Coding Scheme

The microparticles as discussed above have incorporated therein codesderived from any desired coding scheme, such as binary or non-binarycoding.

By way of example, FIG. 39A shows a graphical representation of encodedmicroparticles that are formed according to the invented non-binarycoding scheme. Referring to FIG. 39A, the coding scheme parameters are L(the length of the particle), w (the width of the gap between segments),and d (the delta in the position of the gap center of the gap). FIG. 39Ashows 4 particles with different codes such that only one of the gaps isvaried in location. The gap is varied by amount equal to d, showing“adjacent” codes (e.g. codes that are similar and therefore more likelyto be mis-identified for one another. FIGS. 39B and 39C show randomcodes with different numbers of gaps and gaps of varying location. Table1 presents the total number of codes (codespace) for a variety ofdifferent parameter combinations. The number of codes is calculated froma computer software program that implements the invented non-binarycoding scheme. Code degeneracy is taken into account in the algorithm(e.g. a pair of codes, such that when one is reversed, the codes areequivalent and the two codes are considered a single code). Theparameters in Table 1 and Table 2 are specified in 100 nm units. Theparameter combination L=152, w=8, d=4 which gives 30,069 is shown inFIGS. 39A to 39C. Table 2 presents the total number of codes that can berepresented by the microparticles by different L. In an example, thediscretization distance w is equal to or smaller than the characteristicsegment size. As shown in Table 2, very large codespaces are available,and practically achievable with the aforementioned methods. Theparameter combination L=152, w=5, d=4 has a codespace of approximately 2million.

TABLE 1 Number of Codes L w d (Codespace) 152 8 8 2134 152 8 7 3281 1528 6 5846 152 8 5 11439 152 8 4 30069 152 8 3 105154 100 5 5 3,409 110 55 8,904 120 5 5 23,296 130 5 5 62,376 140 5 5 170,083

TABLE 2 Number of Codes L w d (Codespace) 80 5 4 928 90 5 4 2,683 100 54 7,753 110 5 4 22,409 120 5 4 64,777 130 5 4 187,247 140 5 4 541,252150 5 4 1,564,516 152 5 4 1,934,524 160 5 4 4,522,305

In an example, the coding scheme utilizes code elements placed atlocations spanned by interval lengths smaller than the code element sizeitself. This deviates from the standard binary coding where the codeconsists of the absence or presence of a feature at discrete, evenlyspaced locations. In the preferred embodiment of this coding scheme,naturally applicable to the above structure manufactured using themultiple print technique, the code element is the gap in the segmentedinner opaque material. The gap size is chosen to be one that is reliablydefined by the stepper and photolithography process and also resolvableby the microscope (working at the desired magnification). The gap size,interval length, and particle length determine the codespace (number ofcodes possible). The determination of a codespace involves tradeoffsbetween particle density on the wafer, identification accuracy, opticaldetection system complexity, and particle number per microscope image.Codespaces of over a million can be produced and accurately identifiedusing practical parameter combinations.

In the example of a standard binary coding scheme, the particle would bedivided into units of equal length. Each unit could then be black orwhite, 0 or 1. Because the particle is symmetric, there are two codesthat are the same when one is reversed (so called “degenerate” codes).When counting the codes, one from each of the pair of degenerate codesis preferably discarded. Without the degeneracy, there would be 2^(N)possible codes, where N is the number of bits (units). With thedegeneracy, there are about half that number. Exactly, the number ofpossible codes with the standard binary format is[2^(N)+2^(floor[(N+1)/])]/2. In the example of the high contrast encodedmicroparticle structures of the present invention, previously shown inFIG. 14, FIG. 17, etc., within the full set of codes, there may beindividual codes that have long runs of black or white regions. Theblack of the particles is indistinguishable from the black of thebackground, giving the particles extremely high contrast. However, codeshaving long runs of black are less desirable (though certainly withinthe scope of the invention) because it is more difficult to associatethe white regions into the separate particles. For example, a moredifficult code would be 1000 . . . 0001 (single white bits at bothends). It should be noted that, particularly for the structures andmethods of making mentioned earlier herein, any suitable coding schemecan be used, as many other coding schemes are possible beyond thatdiscussed in the example above.

The non binary coding scheme mentioned above has many advantages in thefabrication and detection of microparticles, including providing forhigh codespaces and robust code identification. In the example of thecoding scheme, the reliability of the microparticle fabrication processis improved by permitting optimization of patterning and etch conditionsfor features, of a single size, e.g. gaps in the segments having asingle width.

In the examples of encoded microparticles and methods of determiningcodes therein, e.g. as shown in FIG. 21, the code is determined by thecenter location of the gaps and not the lengths of segments. Therefore,if the dimensions change, either because of variation in the manufactureor variation in the imaging conditions or variation in the imageprocessing algorithm used, the center position of the gaps does notchange, rendering the code ID is robust. This scheme exploits the factthat in an optical imaging system the position of features, in this casethe gaps, can be located to a resolution much smaller than the minimumresolvable dimension of the features themselves. For example, if the gapwidth may be 1.5 μm or less, and located to a distance smaller than 1.0um, more preferably smaller than 0.5 μm.

In general, a high codespace is desirable. In the field of genomics,having a codespace in the tens of thousands is especially importantbecause it enables full genomes of complex organisms, such as the humangenome, to be placed on a single particle set. The top portion of Table1 shows the effect of varying the delta parameter, d, on the codespace.Shrinking d gives many more codes but places increased demand on theoptical system. The need to resolve a smaller d means that a moreexpensive objective would typically be used. Practically, the lowerlimit of the gap interval distance is set by the resolution by theoptical system (manifested as the pixel size of the digital imagecaptured using a CCD camera). Using a 60× objective and 6.2 mm 1024×1024CCD chip, an interval distance of d=0.4 μm equals approximately 4pixels. If the interval distance is reduced to 0.3 nm (3 pixels), thereare 105,154 codes. The codespace can be extended into the millions forlonger particle lengths, L, and/or smaller gap widths, w.

The lower portion of Table 1 shows the effect of varying the length ofthe particle at fixed w and d. The length L is inversely proportional tothe density of particles on the die (number of particles per unit area).The length also affects the number of particles in an image and thusthroughput (particles detected per second). Tradeoffs exist betweencodespace, density, identification, and throughput. Optimization of thecoding scheme parameters will determine the selected coding scheme for aparticular application.

Large Particle Sets

FIG. 40 shows photographs a montage of 4 photographs of various forms ofa large prototype set of microparticles. The set contains over 1,000codes and approximately 2 million particles of each code. The upper leftphotograph shows 40 wafers during the fabrication process. Each waferhas 32 dies with each die comprising approximately 2 million particlesof a single code. As a further example, dies on a wafer may contain manymore particles per wafer, e.g. 5 million or more. Also, wafers (or othersubstrates, such as glass panels), may contain 100 or more dies, oralternately 200 or more, or 1000 or more dies. The wafer taken in wholemay have 100 or more codes of encoded microparticle, or alternately 200or more, or 1000 or more codes, or 5,000 or more codes. In an example ofa large set of encoded microparticles, substantially all dies used toproduce the large set, e.g. microparticles released from dies, comprisedifferent codes. In another example, all dies on a wafer or substrate,may have the same code. The size of dies may be selected so as tooptimize the balance between the number of particles per code and thenumber of codes in the large set of a large set. The number of particlesper die and dies per wafer may be changed in software, for example byutilizing the invented method of producing codes, and optimized on a permanufacturing lot or per product basis for different applications,without necessitating the high capital costs of fixed tooling, e.g.large and expensive sets of photomasks.

In the upper right photograph of FIG. 40, the wafer fabrication has beencompleted and the particles released from the silicon substrate intotest tubes. The test tubes are shown in the photograph in placed incontainers that each hold 64 test tubes. The photograph in the lowerleft corner shows a single test tube which contains a small portion(approximately a few thousand particles) of each of 1035 test tubes ofparticles from the large set. The lower right image is a microscopeimage of a sample of the single test tube. This image shows members of1035 codes mixed together.

Assays

The encoded microparticles, systems, and methods of the invention have awide range of applications in the fields of biology, chemistry, andmedicine, as well as in security and commercial fields involving thetagging of monetary bills, identification cards and passports,commercial products, and the like. In one example, the microparticlescan be used in for molecular detection, such for as analyzing DNA, RNA,and proteins. In other examples, combinatorial chemistry or drugscreening assays are performed as known in the art.

Referring to the flowchart shown in FIG. 41, microparticles arecontained in separate tubes (or wells of well plates). Each tubecontains a large number (e.g. a million or higher) of microparticles ofa single code, at step 410. Biomolecules, such as DNA or RNA areimmobilized on the surface of the particles and referred to as “probes”at step 412. Each species of probe is immobilized onto a different codeand a lookup table is generated for future reference. Each species ofprobe also has one or more corresponding species of “targets” for whichthe binding between the two is specific. The probe/target terminology isusually used in reference to DNA and RNA complements but in this contextrefers to all biomolecules, including antibodies. Many probes areimmobilized on a single particle, typically with a density on the order10⁴/μm² or higher. The singular use of “a probe” often refers to aplurality of probe molecules; and “a code” often refers to a pluralityof particles of a certain code, as with other terms used herein.

The mating of the encoded particles and biomolecules produces a “pooledprobe set” through step 414. The pooled probe set is a mixture ofencoded particles where each code has a particular probe attached to theparticle surface. The pooled probe set can then be used to determine theamount of individual targets present in a mixture of targets. Themixture of targets is referred to as the sample and is typically derivedfrom a biological specimen. The sample is then labeled, typically with afluorophore at step 416. When the sample is mixed with the pooled probeset, the probes and targets find each other in solution and bindtogether. With nucleic acids, this reaction, step 418, is calledhybridization and is very selective. After the reaction, the particlesare imaged to read the codes and quantify the fluorescence at step 420.Referring to the code-probe lookup table, the amounts of the differenttarget species in the mixed sample can now be measured and as a theassay result determined at step 422.

The samples reacted with the microparticles may be a purified biologicalextract or a non-purified sample, including but not limited to wholeblood, serum, cell lysates, swabs, or tissue extracts. The samplesreacted with the microparticles may be produced by culturing, cloning,dissection, or microdissection. Cells may serve as either the sample orprobe in a bioassay utilizing the microparticles and otheraforementioned inventions.

FIGS. 44 and 45 show dense fluorescence microscope image of amultiplicity of encoded microparticles. The microparticles shown in theimages have oligo probe molecules attached to their surfaces and havebeen hybridized to pre-labeled fluorescent oligo targets, where the basepair sequence of the targets is complementary to the sequence of theprobes.

FIG. 42 shows a diagram of an example of the process by which wholewafers become mixtures of particle-probe conjugates that are ready to bereacted with samples to perform a bioassay (so called“Hybridization-Ready CodeArrays”). After completion of the waferfabrication steps, the wafers have many dies where each die containsmany particles of a single code. As has been previously described,alternative schemes may be used where dies are produced with the samecode or dies are subdivided and contain multiple codes. The wafer isdiced (usually by wafer saw) into the separate dies, then each die isplaced into separate wells of a wellplate. Alternatively, test tubes canbe used instead of wells. A release step is performed e.g. using achemical etchant such as TMAH) that removes the particles from thesurface of the die. The die is then removed from the well, leaving thefree particles. After release, the conjugation of the biomolecule probesis performed resulting in each well containing a single type of particleprobe conjugate (with particles of a single code and those particleshaving a single species of biomolecule on the surface). Afterconjugation, all of the particles are mixed together to form a “pooledmaster mix”. The pooled master mix is divided into aliquots such thatsufficient representation from all species of particle-probe conjugatesis present. These aliquots are then ready to be reacted with a sample toperform a bioassay.

It is noted that multiple different samples may be identified in asingle bioassay as discussed above. Before the detection and after thehybridization, the microparticles can be placed into wells of a wellplate or other container for detection. In one detection example, themicroparticles settle by gravity onto the bottom surface of the wellplate. The microparticles in the well can be subjected tocentrifugation, sonication, or other physical or chemical processes(multiple washing steps, etc.) to assist in preparing the particles fordetection. In another example, the microparticles can be placed onto aglass slide or other specially prepared substrate for detection. In yetother examples, the particles are present in a flow stream duringdetection, or present in a suspended solution.

Term conjugation is used to refer to the process by which substantiallyeach microparticle has one or more probe molecules attached to itssurface. Methods of conjugation are well known in the art, for examplein Bioconjugate Techniques, First Edition, Greg T. Hermanson, AcademicPress, 1996: Part I (Review of the major chemical groups that can beused in modification or crosslinking reactions), Part II (A detailedoverview of the major modification and conjugation chemicals in commonuse today), and Part III (Discussion on how to prepare unique conjugatesand labeled molecules for use in applications).

The molecular probes attached to the surface of the particles typicallyhave known attributes or properties. In an example, the molecular probescan be derived from biological specimens or samples and used in thescreening, including but not limited to genetic sequencing, of largepopulations where typically, the derivatives from one member of thepopulation is applied to a single code, typically a multiplicity ofparticles of a single code. Preferably, microparticles having the samecode have attached substantially the same probe molecules; whereasmicroparticles having different codes likewise have different probemolecules.

One of the most powerful features of a multiplexed assay using solutionarrays of encoded particles as the platform instead of planarmicroarrays is the flexibility to add functionality to the assay bysimply adding new particles. With standard microarrays, once the arraysare printed or synthesized, the array typically cannot be changed. Ifthe researcher wants to change the probes for genes on the array or addprobes for new genes, typically entirely new arrays would then beproduced. With pooled probe sets of particles, new probe and particleconjugates (probes for short) can easily be added to the existing pooledprobe set. In practice the new probes could be different probes for analready represented gene, probes for alternative splicing variants ofgenes, or tiling probes for genes.

FIG. 46A and FIG. 46B show a reflectance and fluorescence image pair forthe same set of microparticles of the invention. The images were takenin succession by about 1 second apart. FIG. 46A, the reflectance imagewas taken with blue light illumination and collection (excitationfilter=436/10 nm, emission filter=457/50, i.e. overlapping filters).This image is used to determine the code of each particle. FIG. 46B, thefluorescence image, was taken with green illumination and red collection(excitation filter=555/28 nm, emission filter=617/73, i.e. filters forCy3). FIG. 46C the image pair of FIG. 46A and FIG. 46B overlaid on topof one another in a single image.

FIG. 47A to FIG. 47F show dense fluorescence microscope images ofencoded microparticles in a time sequence. The images have beenprocessed for edge detection. The images were acquired approximately 1second apart and are frames of the time sequence. The individualparticles that comprise the images move a measurable amount between theframes due to molecular collisions (aka Brownian motion). This Brownianmotion facilitates the assembly of the particles into a dense2-dimensional monolayer. The particles shown in the images are examplesof biochemically active encoded microparticles. The particles haveoligonucleotide probes attached to the surface and have been hybridized(i.e. reacted in solution) with complementary oligonucleotide targets.

A Bioassay Process Using the Microparticle

The microparticles of the invention can be used as major functionalmembers of biochemical (or chemical) analysis systems, including but notlimited to solution based arrays, biochips, DNA microarrays, proteinmicroarrays, lab-on-a-chip systems, lateral flow devices(immunochromatographic test strips). Applications include but are notlimited to gDNA and protein sequencing, gene expression profiling,genotyping, polymorphism analysis, comparative genomic hybridization(CGH), chromatin immunoprecipitation (CHiP), methylation detection, aswell as discovering disease mechanisms, studying gene function,investigating biological pathways, and a variety of other biochemicaland biomolecular related applications such as inspection and analyses ofproteins, peptides, polypeptide, and related biochemical applications.Assay architectures may include those well known in the art, includingbut not limited to direct DNA hybridization, hybridization of DNA to RNAor RNA to RNA, enzymatic assays such as polymerase extension, ligation.The microparticles can also be used in microfluidic or lab-on-a-chipsystems or any flow based systems, including but not limited to thosesystems wherein sample preparation, biochemical reaction, andbio-analyses are integrated.

For example, fluorescent tags can be employed when an optical imagingmethod based on the presence of fluorescence can be used. Radioactivelabels can be used when the microparticles are utilized to expose ordevelop relevant photographic films. Alternatively, enzymatic tags canbe used when the detection involves detection of the product of theenzyme tag that is released when the sample molecules bind to or reactwith the probe molecules on the microparticles. Other tagging methodsare also possible, as set forth in “Quantitative monitoring of geneexpression patterns with a complementary DNA microarray” by Schena etal. Science, 1995, 270-467, the subject matter of which is incorporatedherein by reference in its entirety.

Samples without labels can also be reacted with the microparticles. Forexample, molecular beacon probes can be applied to the microparticle.Molecular beacon probes typically contains a hairpin structure that,upon binding the labelless, or in some examples labeled, samplemolecules unfold, thus producing a signal indicative of the bindingevents. Such molecular beacon probes, as well as other probes, may beused in assays involving FRET (Fluorescence Resonant Energy Transfer),where for example fluorophores or quenchers are placed on or in thesurface of the microparticles.

FIG. 48 shows real assay data from a 2-plex DNA hybridization assay. Inthis experiment, 2 different oligo probes (with 2 different sequencesshown at the bottom) were attached to the surface of the differentparticle batches (with different codes). After probe attachment, theparticles were mixed together and aliquots of the mixture were placedinto two wells of a wellplate. Targets composed of oligos with sequencescomplementary to the probe sequences and fluorophore labels were thenadded to the two wells and reacted with the mixture of particle-probeconjugates. Target 1, complementary to probe 1, was added to the firstwell and target 2, complementary to probe 2, was added to the secondwell. Imaging of the particles of both wells was performed and theresults are shown in FIG. 48. In the first well (with target 1),particles of the corresponding code exhibit a relatively highfluorescence signal, and vice-versa for the second well.

For facilitating fast, reliable, and efficient bioassay for large numberof sample molecules, it is preferred that the microparticles are capableof arranging themselves substantially in a monolayer on a surface, suchas the bottom surface of the well in which the microparticles arecontained. The microparticles are preferred to be able to undergoBrownian motion in the specific liquid in which the optical detection isperformed. Given the specific liquid in which the microparticles arehybridized and detected, it is preferred that the 2D diffusioncoefficient of the microparticles is equal to or greater than 1×10⁻¹²cm²/s and/or 10% or more, such as 15% or more, or even 20% or more, and50% or more of the microparticles are measured to undergo a lateraldisplacement of 20 nm or greater, such as 30 nm or greater, or even 50nm or greater—in a time interval of 1 second or less, or preferably 3seconds or less, or five seconds or less.

The detectable microparticles, which are referred to as those that areable to be accurately detected by the desired detection means, such asoptical imaging using visible light, are capable of occupying 30% ormore, 40% or more, and typically 50% or more of the surface area onwhich the microparticles are collected together, such as a portion ofthe bottom surface of the container in which the microparticles arecontained. Defining an area in which at least 90% of all themicroparticles are disposed (typically at least 95% or more typically atleast 99%, and often 100%), the microparticles can be seen to have adensity of 1000 particles/mm² or more, such as 1500 particles/mm² ormore, 2000 particles/mm² or more, and typically 3000 particles/mm² ormore (e.g. 5000 particles/mm² or more). The detection rate within theabove-mentioned area, which rate is defined as the ratio of the totalnumber of detected microparticles (microparticles with spatial codesdetected) of a collection of microparticles under detection to the totalnumber of the collection of microparticles, is preferably 80% or more,typically 90% or more, or more typically 99% or more.

Another preferred example of the invention is a kit comprisingbiochemically active encoded microparticles that contains 200 or more,more preferably 500 or more, 1000 or more, or even 10,000 or moredifferent codes within the kit (due to the large codespace enabled bythe invention, even larger numbers of codes). Due to statistical samplerequirements of convenient liquid pipetting and a desired redundancy ofparticular codes within the kit, more than 10 particles of the same codeare typically provided (20 or more, or even 30 or more microparticles ofthe same code) within the kit, as in some example applications theredundancy improves the overall assay performance. The term“biochemically active encoded microparticles” is refers tomicroparticles that have biological or chemical moieties on surfaces andthus can be used in assays; and the term “moieties” are referred to asmolecular species; including but are not limited to nucleic acids,synthetic nucleic acids, oligonucleotides, single stranded nucleicacids, double stranded nucleic acids, proteins, polypeptides,antibodies, antigens, enzymes, receptors, ligands, and drug molecules,cells, and complex biologically derived samples.

Universal adapter schemes may be used to provide a set ofnon-interacting synthetic sequences that are complementary to sequencesprovided on the probes. Genotyping can be performed using common probesand allele specific reporters or allele specific probes and commonreporters. Amplification assays such as those involving PCR, padlockprobes, or Molecular Inversion Probes can be performed using theparticles of the current invention. Examples of two of these assays areshown in FIG. 49A and FIG. 49B. In an alternative example of theinvention, biomolecules that are present on the surface of the particlescan be pre-synthesized and then attached to the particle surface.Alternatively, biomolecules can be in situ synthesized on the particles.

The encoded microparticles may have attached thereto manufactured orsynthetic DNA oligonucleotides to allow the described nucleic acid-basedexperiments and assays to be conducted on the encoded microparticles.Because of the manner in which the present encoded microparticles aremanufactured, in large batches, etc., and because they can beuniversally adapted with many different probes after manufacture, andcan be combined in any number of plex, i.e. for multiplex assays, smallscale assays and applications may be designed very inexpensively andefficiently. The encoded microparticles may be stored for long periodsof time and simply combined “off the shelf” to make as small or as largean assay as may be desired and in as many different plex as desired.

Furthermore, the encoded microparticles in small-scale experiments couldbe used in assays that employ cloned or PCR-generated cDNA sequences,instead of manufactured oligonucleotides. Further, positive and negativecontrols may be synthesized as probes and thus small-scale assays may bedesigned which employ both cloned and/or PCR-generated oligonucleotideprobes as well as synthetic probes in a mixture.

For instance, the user may provide the cDNA generated in a typicalmanner in which, for instance, an purified mRNA from a cell culture isreverse transcribed to create a certain amount of cDNA. These cDNA maythen be attached to the encoded microparticles to function as the probesin the “liquid array” type of assay.

Protein based assays are also applicable. These include but are notlimited to sandwich immunoassays, antibody-protein binding assays,receptor-ligand binding assays, or protein-protein interaction assays.Examples of these assays are shown in FIG. 50. The sets of encodedmicroparticles of the present invention can be used in solution basedassays to investigate protein-protein interactions. This is shown in thebottom right of FIG. 50.

A single type of protein can be applied to microparticles of a singlecode. Upon mixing of the particle-protein conjugates and reaction in aparticular biochemical environment, proteins that interact and bind toone another are determined by the presence of adjacent particles duringdetection. The square cross section of the microparticle structures ofthe present invention provide an improvement over the prior art byproviding an increased area of contact in the shape of a flat,rectangular surface. Prior art particles that are spherical orcylindrical in shape limit the contact areas to single points or linesrespectively. This invention is not limited to proteins: any interactingmolecules may be used with this assay architecture. Also, theomni-directional encoded microparticles of the present invention may beused in conjunction with any other encoded particles including but notlimited to fluorophores, quantum dots, latex or glass beads, colloidalmetal particles, spectroscopically active particles, SERS particles, orsemiconductor nanorods.

The encoded microparticles may be used in conjunction with a 2D planararray of molecules. Interaction between molecules on the surface of theparticles and those contained in spots on the 2D planar array aredetermined by the binding of the particles to the spots. The presence ofthe particles in the predetermined spot locations, preferably afterwashing steps, indicates a binding interaction between the molecules onthe particles and the molecules on the 2D planar array. The assay resultcan be determined by identifying 1) the particle code, and 2) the spotlocation. This is shown in FIG. 51. FIG. 51 is a schematic that includesimages of particles but is not the result of an actual experiment, i.e.meant to serve as an illustration of this invention. In this invention,the square cross section of the microparticles of the present inventionprovide for increased binding contact area and is a significantimprovement over the prior art.

The microparticles of the invention may have other applications. Forexample, by placing protein-detection molecules (e.g., ligands, dyeswhich change color, fluoresce, or cause electronic signal upon contactwith specific protein molecules) onto the microparticles, bioassayanalyses can be performed (i.e., evaluation of the protein and/or geneexpression levels in a biological sample). As another example, byplacing (cellular) receptors, nucleic acids/probes, oligonucleotides,adhesion molecules, messenger RNA (specific to which gene is “turned on”in a given disease state), cDNA (complementary to mRNA coded-for by eachgene that is “turned on”), oligosaccharides & other relevantcarbohydrate molecules, or cells (indicating which cellular pathway is“turned on”, etc.) onto the microparticles, the microparticles can beused to screen for proteins or other chemical compounds that act againsta disease (i.e., therapeutic target); as indicated by (the relevantcomponent from biological sample) adhesion or hybridization to specificspot (location) on the microarray where a specific (target molecule) wasearlier placed/attached. In fact, the microparticles of the inventioncan be applied to many other biochemical or biomolecular fields, such asthose set forth in the appendix attached herewith, the subject matter ofeach is incorporated herein by reference.

It will be appreciated by those of skill in the art that a new anduseful microparticle and a method of making the same have been describedherein. The large sets of encoded microparticles produced by thisinvention can be a fundamental technology that will have far reachingapplications, especially in the field of biotechnology and morespecifically genomics. It has the potential to dramatically reduce thecost of highly multiplexed bioassays. Moreover, enables researchers toeasily design custom content solution arrays. The researcher can alsoeasily add new particle types to the pooled set, for instance includingnew found genes of interest with the microparticles of the invention.

In view of the many possible embodiments to which the principles of thisinvention may be applied, however, it should be recognized that theembodiments described herein with respect to the drawing figures aremeant to be illustrative only and should not be taken as limiting thescope of invention. Those of skill in the art will recognize that theillustrated embodiments can be modified in arrangement and detailwithout departing from the spirit of the invention.

For example, the microparticle may have a six sided shape with fourelongated sides and two end sides. The encoded microparticle can beconfigured such that the code of the encoded microparticle can bedetectable regardless of which of the four elongated sides the barcodeis disposed on. The microparticle may have a ratio of the length towidth is from 2:1 to 50:1, from 4:1 to 20:1. The length of themicroparticle is preferably from 5 to 100 μm and more preferably lessthan 50 μm. The width of the microparticle can be from 0.5 to 10 μm. Inother examples, the length of the microparticle can be less than 10 um,less than 25 um, less than 25 um; less than 5 um, less than 27 um; andthe width of the microparticle can be less than 3 μm. The ratio of widthto height of the microparticle can be from 0.5 to 2.0. The ratio of thelength to width of the microparticle can be from 2:1 to 50:1. The crosssection taken along the length of the microparticle is substantiallyrectangular with a length at least twice the width.

The microparticle may have a glass body with segments embedded therein.The difference of the transmissivity of the glass body and segments canbe 10% or more. The glass body may have a length of less than 50 μm anda width of less than 10 μm with the glass body having a volume of from 5to 500 μm³. The encoded microparticle may have 2 to 15, 3 to 10, or 4 to8 portions of less transparent material within the encodedmicroparticle. The code incorporated in the microparticle can be binaryor non-binary or any other desired codes. The microparticle may havebiochemical molecules attached to one or more surfaces of themicroparticle, such as DNA and RNA probes with a density of from 10² to10⁶/μm². When fabricated on the wafer-level, the wafer may have asurface area of from 12.5 in² to 120 in², and wherein there are at least3 million microparticles per in² of the wafer. The wafer may have atleast one million codes are formed on the substrate, or at least twohundred different codes are present within the one million codes, or atleast 3000 different codes are present within the one million codes.When placed in a liquid buffer, for example in a bioassay, themicroparticles can form a single monolayer with a 2 dimensionaldiffusion coefficient of the microparticles greater than 1×10⁻¹² cm²/sand more preferably greater than 1×10⁻¹¹ cm²/s.

Therefore, the invention as described herein contemplates all suchembodiments as may come within the scope of the following claims andequivalents thereof.

1. A method of forming a plurality of encoded microparticles comprisingthe steps of: (a) providing a planar substrate on which the encodedmicroparticles are to be formed; (b) providing a reticle having areticle pattern; (c) exposing the substrate to light transmitted throughthe reticle, thereby printing a first portion of an encoding patternonto the substrate; (d) moving the substrate in a lateral direction; (e)exposing the substrate to light transmitted through the reticle, therebyprinting the reticle pattern onto the substrate; (f) optionallyrepeating steps (d) and (e) to create the encoding pattern; and (g)removing a portion of the remaining substrate to form the encodedmicroparticles.
 2. The method according to claim 2, wherein the planarsubstrate comprises at least one magnetic layer.
 3. The method accordingto claim 2, wherein the magnetic layer is selected from the groupconsisting of: magnetic, ferromagnetic, diamagnetic, paramagnetic andsuperparamagnetic.
 4. The method according to claim 2, wherein themagnetic material is selected from the group consisting of: nickel,cobalt, and iron.
 5. The method according to claim 2, wherein the layeris comprised of ferromagnetic nanoparticles.
 6. The method according toclaim 5, wherein the ferromagnetic nanoparticles are layered onto thesubstrate using a spin-on-glass (SOG) technique.
 7. The method accordingto claim 6, wherein the encoded microparticles are superparamagnetic. 8.The method according to claim 7, wherein the encoded microparticles donot attract one another due to their magnetic properties.
 9. The methodaccording to claim 1, wherein the planar substrate comprises at leasttwo magnetic layers, and wherein at least one of the at least twomagnetic layers is comprised of ferromagnetic nanoparticles.
 10. Themethod according to claim 2, further comprising attaching a plurality ofoligonucleotides to the surface of the microparticles.
 11. The methodaccording to claim 2, wherein the method employs photolithography.
 12. Amethod of detecting a false positive encoded microparticle in abiological assay, which comprises: providing one or more encodedmicroparticles resting on a clear well plate through which the one ormore encoded microparticles may be visualized, wherein one or more ofthe one or more encoded microparticles is labeled, and wherein the oneor more encoded microparticles comprise: a longest dimension less than50 microns; a plurality of segments, wherein the plurality of segmentsform a spatial code; and an outer surface, wherein the outer surfaceencloses the spatial code, and wherein the spatial code is detectablethrough the outer surface; storing a digital image of the one or moreencoded microparticles; determining a first, second and third opticalapertures around each visualized encoded microparticle image; measuringlabel signal intensity within the first, second and third opticalapertures, wherein if the label signal intensity increases from thefirst optical aperture to the third optical aperture it is determined tobe a false positive encoded microparticle, thereby detecting a falsepositive encoded microparticle.
 13. The method according to claim 12,wherein the digital image is captured by scanning with a CCD camera. 14.The method according to claim 12, wherein the one or more labeledencoded microparticles is labeled with a fluorescent dye because the oneor more labeled encoded microparticles specifically bound to one or moretargets.
 15. The method according to claim 12, wherein the one or moreencoded microparticles have attached thereto one or moreoligonucleotides.
 16. The method according to claim 15, wherein the oneor more encoded microparticles are incubated with a sample comprising orsuspected of comprising one or more labeled targets.
 17. The methodaccording to claim 15, wherein the one or more oligonucleotides weregenerated by reverse PCR of a sample comprising one or more mRNA. 18.The method according to claim 15, wherein the one or moreoligonucleotides are optionally labeled with one of a FRET label pair.19. The method according to claim 12, wherein the first, second andthird optical apertures are automatically defined in a two-dimensionalspace around each encoded microparticle by a computer software programresiding in a computer.
 20. The method according to claim 19, wherein auser may define where the first, second and third apertures are locatedaround the encoded microparticles.
 21. The method according to claim 12,wherein a volume of the one or more encoded microparticles is 20,000cubic microns or less.